Vaccines and compositions against streptococcus pneumoniae

ABSTRACT

Streptococcus pneumoniae  is a major health concern, especially in very young, elderly, or immunocompromised patients. The present disclosure provides, inter alia, certain highly effective vaccines and pharmaceutical compositions in  Streptococcus pneumoniae . The antigens may be used therapeutically or prophylactically.

RELATED APPLICATIONS

This application claims the benefit of the filing date of U.S.Provisional Application No. 61/221,541, filed Jun. 29, 2009, U.S.Provisional Application No. 61/240,616, filed Sep. 8, 2009, U.S.Provisional Application No. 61/240,598, filed Sep. 8, 2009, and U.S.Provisional Application No. 61/316,267, filed Mar. 22, 2010. The entireteachings of the referenced applications are expressly incorporatedherein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Jun. 28, 2010, is named559231US.txt and is 142,908 bytes in size.

I. BACKGROUND

Pneumococcal disease continues to be a leading cause of sickness anddeath in the United States and throughout the world. Each year, millionsof cases of pneumonia, meningitis, bacteremia, and otitis media areattributed to infection with the pathogen Streptococcus pneumoniae. S.pneumoniae is a Gram-positive encapsulated coccus that colonizes thenasopharynx in about 5-10% of healthy adults and 20-40% of healthychildren. Normal colonization becomes infectious when S. pneumoniae iscarried into the Eustachian tubes, nasal sinuses, lungs, bloodstream,meninges, joint spaces, bones and peritoneal cavity. S. pneumoniae hasseveral virulence factors that enable the organism to evade the immunesystem. Examples include a polysaccharide capsule that preventsphagocytosis by host immune cells, proteases that inhibitcomplement-mediated opsonization, and proteins that cause lysis of hostcells. In the polysaccharide capsule, the presence of complexpolysaccharides forms the basis for dividing pneumococci into differentserotypes. To date, 93 serotypes of S. pneumoniae have been identified.

Various pharmaceutical compositions have been used to harness an immuneresponse against infection by S. pneumoniae. A polyvalent pneumococcalvaccine, PPV-23, was developed for preventing pneumonia and otherinvasive diseases due to S. pneumoniae in the adult and agingpopulations. The vaccine contains capsular polysaccharides (CPs) from 23serotypes of S. pneumoniae. As T cell independent antigens, these CPsinduce only short-lived antibody responses, necessitating repeateddoses, which increases the risk of immunological tolerance. Theantibodies raised against S. pneumoniae, termed anticapsular antibodies,are recognized as protective in adult and immunocompetent individuals.However, children under 2 years of age and immunocompromisedindividuals, including the elderly, do not respond well to T-cellindependent antigens and, therefore, are not afforded optimal protectionby PPV-23. A second S. pneumoniae vaccine, Prevnar, includes bacterialpolysaccharides from 7 S. pneumoniae strains conjugated to thediphtheria toxoid protein. This vaccine induces both B and T cellresponses. However, because it only protects against 7 pneumococcalserotypes, serotype replacement can render Prevnar ineffective. PPV-23suffers from the same limitation. Serotype replacement has already beendemonstrated in several clinical trials and epidemiologic studies, andraises the possibility that different formulations of the vaccines willneed to be developed, presumably at even higher cost. Furthermore, bothPPV-23 and Prevnar are expensive to manufacture, greatly limiting theiravailability in the developing world.

Thus, there remains a need to design more effective pharmaceuticalcompositions than the current strategies offer. In particular, suchcompositions need to incorporate novel or specific antigens that elicitan immune response against S. pneumoniae.

II. SUMMARY

Streptococcus pneumoniae is a major health concern, especially in veryyoung, elderly, or immunocompromised patients. While DNA and proteinsequence information for S. pneumoniae has been known for some time, andresearchers have long attempted to produce vaccines against S.pneumoniae, a major problem was how to identify protective polypeptidesfrom among the approximately 2100 genes in the S. pneumoniae genome. Theinstant application presents the results of whole-genome screensdesigned to identify the most immunogenic proteins in the S. pneumoniaegenome. Several of the hits from the screen have been shown to protectagainst S. pneumoniae colonization in a mouse model. Accordingly, thepresent disclosure provides, inter alia, certain highly effectivevaccines in Streptococcus pneumoniae. The vaccines may be usedtherapeutically or prophylactically. The present disclosure alsoprovides specific antigens and methods for using the antigens to elicitan immune response against S. pneumoniae.

The present disclosure provides, for example, a vaccine formulationcomprising a pharmaceutically acceptable carrier and one or morepolypeptides having an amino acid sequence comprising any of SEQ ID NOS:1-11 or an immunogenic fragment thereof, and optionally furthercomprising a polypeptide having an amino acid sequence comprising eitherof SEQ ID NOS: 12 or 13 or an immunogenic fragment thereof.

The present disclosure also provides a vaccine formulation comprising apharmaceutically acceptable carrier and a polypeptide having an aminoacid sequence consisting of SEQ ID NO: 11 or an immunogenic fragmentthereof. In addition, the present disclosure provides a vaccineformulation comprising a pharmaceutically acceptable carrier and apolypeptide having an amino acid sequence comprising SEQ ID NO: 12.

Furthermore, the instant application provides a vaccine formulationcomprising a pharmaceutically acceptable carrier and one or morepolypeptides having an amino acid sequence comprising any of SEQ ID NOS:14-21 or an immunogenic fragment thereof.

This application provides, inter alia, a method for treating a subjectsuffering from or susceptible to S. pneumoniae infection, comprisingadministering an effective amount of any of the vaccine formulationsdescribed herein.

The present disclosure further provides an immunogenic compositioncomprising a pharmaceutically acceptable carrier and two or morepolypeptides having amino acid sequences comprising any of SEQ ID NOS:1-13 or an immunogenic fragment thereof, wherein at least one of saidpolypeptides has an amino acid sequence comprising one of SEQ ID NOS:1-10 or an immunogenic fragment thereof.

III. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the concentration of IL-17 generated by blood samples frommice that were immunized with the indicated protein(s) and choleratoxin, then stimulated with killed, unencapsulated whole cell S.pneumoniae, as described in Example 5. The left panel shows the data inscatter format, and the right panel shows the average and standarddeviation for each sample Immunization group “All 3” represents animalsimmunized with a combination of SP2108, SP0148, and SP1634.

FIG. 2 shows the concentration of IL-17 generated by blood samples frommice that were immunized with the indicated protein(s) and choleratoxin, then stimulated with a combination of three proteins (SP2108,SP0148, and SP1634), as described in Example 5.

FIG. 3 shows the number of S. pneumoniae colonies obtained from a nasalwash in mice that were immunized with the indicated protein(s) andcholera toxin, then challenged with intranasal administration of S.pneumoniae, as described in Example 5. 003 represents a controlirrelevant antigen.

FIG. 4 shows the concentration of IL-17 generated by blood samples frommice that were immunized with the indicated protein(s) and choleratoxin, then stimulated with killed, unencapsulated whole cell S.pneumoniae, as described in Example 6.

FIG. 5 shows the concentration of IL-17 generated by blood samples frommice that were immunized with the indicated protein(s) and choleratoxin, then stimulated by the indicated proteins and combination, asdescribed in Example 5.

FIG. 6 shows the number of S. pneumoniae colonies obtained from a nasalwash in mice that were immunized with the indicated protein(s) andcholera toxin, then challenged with intranasal administration of S.pneumoniae, as described in Example 6. The HSV-2 protein ICP47 with thegene name US12 (NP_(—)044543.1, NC_(—)001798.1; shown in the figure as003) and ovalbumin (OVA) represent control antigens.

FIG. 7 shows the number of S. pneumoniae colonies obtained from a nasalwash in mice that were immunized with the indicated protein(s) andcholera toxin, then challenged with intranasal administration of S.pneumoniae, as described in Example 7.

FIG. 8 shows the number of S. pneumoniae colonies obtained from a nasalwash in BALB/c mice that were immunized with the indicated protein(s)and cholera toxin, then challenged with intranasal administration of S.pneumoniae, as described in Example 8.

IV. DETAILED DESCRIPTION A. Specific Polypeptides and Nucleic Acids forUse in S. pneumoniae Vaccines and Immunogenic Compositions

This application describes S. pneumoniae vaccines that include one ormore of the polypeptides or genes listed in Table 1, or variants orfragments thereof as described below. The vaccine may include apolypeptide that comprises a sequence of Table 1 or a variant orimmunogenic fragment thereof or a polypeptide that consists of asequence of Table 1 or a variant or immunogenic fragment thereof. TheDNA and protein sequence of each gene and polypeptide may be found bysearching for the Locus Tag in the publicly available database, EntrezGene (on the NCBI NIH web site on the World Wide Web, at www.ncbi.nlmnih.gov/sites/entrez?db=gene), in the Streptococcus pneumoniae TIGR4genome, and the indicated sequences are also included in thisapplication.

TABLE 1 Immunogenic polypeptides for vaccine formulations Protein DNADNA GenBank SEQ ID SEQ ID Accession No. Locus tag name and descriptionNo. No. (from Mar. 30, 2010) SP0024 1 — NC_003028.3|:27381-27878 SP08822 — NC_003028.3|:831804-832628 SP0882N 3 24 — SP0882 with exogenousleader 4 25 — SP0882N with exogenous leader 5 26 — SP0148 lacking signalsequence 6 27 — SP0148 including signal sequence 7 28 SP1072 8 —NC_003028.3|:1008420-1010180 SP2108 including signal sequence 9 —NC_003028.3|:2020750-2022021 SP2108 lacking signal sequence 10 29 —SP0641M 11 30 — SP0641 12 — NC_003028.3|:2020750-2022021 SP0641N 13 31 —SP0882 consensus 14 — — SP0882N consensus 15 — — SP0882 consensus withexogenous 16 — — leader SP0882N consensus with exogenous 17 — — leaderSP0148 consensus lacking signal 18 — — sequence SP0148 consensusincluding signal 19 — — sequence SP2108 consensus lacking signal 20 — —sequence SP2108 consensus including signal 21 — — sequence SP1634 22 —NC_003028.3|:1534348-1535421 SP0314 23 — NC_003028.3|:287483-290683

Certain polypeptides of Table 1, and variants thereof, are described ingreater detail below.

1. SP0024 (SEQ ID NO: 1) and Variants Thereof.

SP0024 represents a hypothetical protein of 165 amino acids, containinga conserved carbonic anhydrase domain that extends from amino acid 27 toamino acid 163. Based on this consensus motif, SP0024 may be azinc-binding protein.

In some embodiments, vaccines or pharmaceutical compositions comprisingan S. pneumoniae polypeptide include a polypeptide containing at least20 consecutive amino acid residues selected from SP0024. The polypeptidemay also be a variant of the at least 20 residue fragment. In certainembodiments, the polypeptide includes no more than 150, 125, or 100consecutive amino acids from SP0024.

2. SP0882 (SEQ ID NO: 2) and Variants Thereof.

SP0882 is a conserved hypothetical protein of 274 amino acids. Much ofthe protein (amino acids 2-270) forms an esterase or lipase-like region.

In some embodiments, vaccines or pharmaceutical compositions comprisingan S. pneumoniae polypeptide include a polypeptide containing at least20 consecutive amino acid residues selected from SP0882. The polypeptidemay also be a variant of the at least 20 residue fragment. In certainembodiments, the polypeptide includes no more than 250, 275, 200, 175,150, 125, or 100 consecutive amino acids from SP0882.

One particular truncation variant named SP0882N consists of theN-terminal 130 amino acids of SP0882, and is shown as SEQ ID NO: 3.SP0882N includes a region that is particularly well conserved amongdifferent serotypes. In certain embodiments, a polypeptide comprisingSP0882 or SP0882N, or an immunogenic fragment of either, also comprisesan exogenous leader sequence. The leader sequence may be, for example,the leader sequence of SP2108. Two exemplary such polypeptides are SEQID NOS: 4 and 5.

Variants of DNA and protein sequences of SP0882 are described, interalia, in US Patent Application Publication No. 2009/0215149 andInternational Applications WO2002/077021, WO98/18931, and WO2007/106407.A variant of SP0882N is disclosed in International ApplicationWO2008/146164.

Sequence variation occurs at the protein level between different S.pneumoniae serotypes, and consensus sequences illustrating combinationsof SP0882 sequences from different S. pneumoniae serotypes are providedas SEQ ID NOS: 14-17. Accordingly, in certain embodiments, the vaccineformulation comprises a polypeptide having an amino acid sequencecomprising, or consisting of, any of SEQ ID NOS: 14-17, or animmunogenic fragment thereof (e.g., in place of a polypeptide having anamino acid sequence comprising one of SEQ ID NOS: 2-5).

Nucleic acid sequences encoding different variants of SP0882 areprovided as SEQ ID NOS: 24-26, although due to degeneracy in the geneticcode, other DNA sequences (including codon-optimized sequences) couldencode these polypeptides.

3. SP0148 (SEQ ID NO: 7) and Variants Thereof.

The protein SP0148 is named “ABC transporter, substrate-bindingprotein”. Proteins of this class are typically extracellular proteinsthat interact transiently with a transmembrane protein complex. Suchcomplexes use energy generated by ATP hydrolysis to translocate specificsubstrates across a cell membrane. SP0148 is a 276 amino acid proteinthat contains a conserved PBPb (periplasmic binding protein) domain,spanning amino acids 40-246, which is typical of membrane-boundtransport complexes. In addition, SB0148 has a bacterial extracellularsolute-binding proteins, family 3 domain which is largely co-extensivewith the PBPb domain and extends from amino acid 40 to 244. In someembodiments, a vaccine or other composition comprises a truncationmutant of SP0148 comprising or lacking one or more of said domains andmotifs.

In some embodiments, vaccines or pharmaceutical compositions comprisingan S. pneumoniae polypeptide include a polypeptide containing at least20 consecutive amino acid residues selected from SP0148. The polypeptidemay also be a variant of the at least 20 residue fragment. In certainembodiments, the polypeptide includes no more than 250, 275, 200, 175,150, 125, or 100 consecutive amino acids from SP0148.

Endogenous SP0148 comprises a putative signal sequence that may directits secretion. In some embodiments, a variant of SP0148 that lacks thesignal sequence (SEQ ID NO: 6) is used. The polypeptide of SEQ ID NO: 6is encoded by the nucleic acid of SEQ ID NO: 27, although other nucleicacid sequences (including codon-optimized sequences) may be used. SEQ IDNO: 28 encodes the full length sequence of SP0148 used in the screensherein.

Variants of the amino acid sequence and nucleotide sequence of SP0148may be found in U.S. Patent Application Publication No. 2005/0020813,U.S. Pat. Nos. 7,378,514 and 7,504,110, and European Patent ApplicationNo. EP1572868 and EP1855717.

Consensus sequences illustrating combinations of SP0148 sequences fromdifferent S. pneumoniae serotypes are provided as SEQ ID NOS: 18 and 19.Accordingly, in certain embodiments, the vaccine formulation comprises apolypeptide having an amino acid sequence comprising, or consisting of,either of SEQ ID NOS: 18-19, or an immunogenic fragment thereof (e.g.,in place of a polypeptide having an amino acid sequence comprising oneof SEQ ID NOS: 6 or 7).

4. SP1072 (SEQ ID NO: 8) and Variants Thereof.

SP1072, also known as dnaG, is a DNA primase enzyme that catalyzesformation of an RNA primer which allows DNA polymerase to initiate DNAreplication. A protein of 586 amino acids, SP1072 contains severalconserved motifs. Beginning at the N-terminus, amino acids 2-96 form azinc finger domain, the DNA primase catalytic core spans amino acids122-250, and a highly conserved topoisomerase-primase (TORPIM)nucleotidyl transferase/hydrolase domain region extends from amino acid258 to 330. In some embodiments, a vaccine or other compositioncomprises a truncation mutant of SP1072 comprising or lacking one ormore of said domains and motifs.

In some embodiments, vaccines or pharmaceutical compositions comprisingan S. pneumoniae polypeptide include a polypeptide containing at least20 consecutive amino acid residues selected form SP1072. The polypeptidemay also be a variant of the at least 20 residue fragment. In certainembodiments, the polypeptide includes no more than 550, 500, 450, 400,350, 300, 250, 200, 150, or 100 consecutive amino acids from SP1072.

5. SP2108 (SEQ ID NO: 9) and Variants Thereof.

The polypeptide SP2108 is 423 amino acids in length and is alternativelyknown as MaIX, maltose/maltodextrin ABC transporter, ormaltose/maltodextrin-binding protein. Much of the protein (amino acids3-423) is classified as a MalE (Maltose-binding periplasmic) domain. Inaddition, SP2108 contains a signal sequence that directs its secretion.In some embodiments, a vaccine or other composition comprises atruncation mutant of SP2108 comprising one or more of said domains andmotifs.

In some embodiments, the compositions and methods herein call for theuse of an SP2108 variant that lacks the signal sequence. This variant isrepresented by polypeptide sequence SEQ ID NO: 10 and may be encoded by,for example, a nucleic acid according to SEQ ID NO: 29.

In some embodiments, vaccines or pharmaceutical compositions comprisingan S. pneumoniae polypeptide include a polypeptide containing at least20 consecutive amino acid residues selected from SP2108. The polypeptidemay also be a variant of the at least 20 residue fragment. In certainembodiments, the polypeptide includes no more than 400, 350, 300, 250,200, 150, or 100 consecutive amino acids from SP2108.

Consensus sequences illustrating combinations of SP2108 sequences fromdifferent serotypes are provided as SEQ ID NOS: 20 and 21. Thus, incertain embodiments, the vaccine formulation comprises a polypeptidehaving an amino acid sequence comprising, or consisting of, either ofSEQ ID NOS: 20-21, or an immunogenic fragment thereof (e.g., in place ofa polypeptide having an amino acid sequence comprising one of SEQ IDNOS: 9 or 10).

6. SP0641 (SEQ ID NO: 12) and Variants Thereof

At 2144 amino acids in length, SP0641 is also known as PrtA, a cellwall-associated serine protease. Full-length SP0641 contains a number ofconserved motifs: the PA_(—)2 motif, extending between amino acids 485and 597, which may form a protein binding surface; the Fn3-like domain(amino acids 800-939); and two predicted catalytic domains of the S8 C5atype located at amino acids 226-449 and 639-777. In some embodiments, avaccine or other composition comprises a truncation mutant of SP0641comprising or lacking one or more of said domains and motifs.

In some embodiments, vaccines or pharmaceutical compositions comprisingan S. pneumoniae polypeptide include a polypeptide containing at least20 consecutive amino acid residues selected from SP0641. The polypeptidemay also be a variant of the at least 20 residue fragment. In certainembodiments, the polypeptide includes no more than 1000, 900, 800, 700,600, 500, 400, 300, 200, or 100 consecutive amino acids from SP0641.

Certain other truncation mutants of SP0641 may also be used. Forinstance, the polypeptide designated SP0641N (SEQ ID NO: 13) consists of661 amino acids corresponding to amino acids 24-684 near the N-terminusof SP0641. Roughly adjacent to SP0641N (and corresponding to amino acids686-1333 of SP0641) lies the 648 residue region captured by thetruncation variant SP0641M (SEQ ID NO: 11).

Variants of SP0641 are disclosed in, for example, U.S. Pat. Nos.7,338,786, 6,573,082, and 7,132,107, as well as InternationalApplication WO00/06738.

SEQ ID NOS: 30 and 31 display the DNA sequences of SP0641M and SP0641N,respectively, although due to degeneracy in the genetic code, other DNAsequences (including codon-optimized sequences) could encode SP0641.

Polypeptides homologous to the polypeptides of Tables 1 and 2 (forexample, SP0024, 0882, 0882N, 0148 with or without a signal sequence,1072, SP1028 with or without a signal sequence, SP0641, SP0641M, orSP0641N) may also be used in the compositions and methods disclosedherein. Individual strains of S. pneumoniae contain numerous mutationsrelative to each other, and some of these result different proteinsequences between the different strains. One of skill in the art mayreadily substitute an amino acid sequence, or a portion thereof, withthe homologous amino acid sequence from a different S. pneumoniaestrain. In certain aspects, this application provides immunogenicpolypeptides with at least 90%, 95%, 97%, 98%, 99%, or 99.5% identity tothe polypeptides of Tables 1 and 2 or an immunogenic fragment thereof.Serotypic variation may be used to design such variants of thepolypeptides of Tables 1 and 2.

In some embodiments, the vaccine compositions herein comprise a fragmentof a protein of Table 1 or 2 (for example, fragments of SP0024, SP0882,SP0882N, OSP148 with or without a signal sequence, SP1072, SP1028 withor without a signal sequence, SP0641, SP0641M, or SP0641N). In someembodiments, this application provides truncation mutants that are closein size to the polypeptide of Table 1 or 2 (for example, one of SEQ IDNOS: 1-13). For example, they may lack at most one, two three, four,five, ten, or twenty amino acids from one or both termini Internaldeletions, e.g., of 1-10, 11-20, 21-30, or 31-40 amino acids, are alsocontemplated.

In certain embodiments the vaccine formulation comprises one or morepolypeptides having an amino acid sequence comprising, or consisting of,any of SEQ ID NOS: 14-21. In certain embodiments, the fragment is atruncated fragment of any of SEQ ID NOS: 14-21 wherein from 1-5, 1-10,or 1-20 amino acid residues are removed from the N-terminus, C-terminus,or both. In certain embodiments, the fragment is a truncated fragment ofany of SEQ ID NOS: 14-21 wherein from 1-10 amino acid residues areremoved from the N-terminus, C-terminus, or both. For instance, 10 aminoacid residues may be removed from each of the N-terminus and C-terminusresulting in a protein with 20 amino acid residues removed.

In addition to those nucleic acids and polypeptides described in Table 1above, this application also provides immunogenic compositions thatinclude one or more of the polypeptides or genes listed in Table 1and/or Table 2, or variants or fragments thereof as described herein.The DNA and protein sequence of each gene and protein may be found bysearching for the Locus Tag in the publicly available database, EntrezGene, as described above.

TABLE 2 Immunogenic proteins identified in human and mouse screens Locustag Protein accession DNA accession number (from name number Mar. 30,2010) SP1574 AAK75660.1 NC_003028.3|:c1481367-1480609 SP1655 AAK75734.1NC_003028.3|:c1557922-1557230 SP2106 AAK76165.1NC_003028.3|:c2018657-2016399 SP1473 AAK75567.1NC_003028.3|:c1386534-1386277 SP0605 AAK74757.1NC_003028.3|:571604-572485 SP1177 AAK75286.1NC_003028.3|:c1115580-1115317 SP0335 AAK74510.1NC_003028.3|:306559-306876 SP0906 AAK75031.1 NC_003028.3|:c859160-859029SP1828 AAK75901.1 NC_003028.3|:c1740010-1739000 SP2157 AAK76211.1NC_003028.3|:c2072146-2070995 SP1229 AAK75335.1NC_003028.3|:c1163388-1161718 SP1128 AAK75238.1NC_003028.3|:1061773-1063077 SP1836 AAK75909.1NC_003028.3|:1746104-1746280 SP1865 AAK75937.1NC_003028.3|:c1772987-1771923 SP0904 AAK75029.1NC_003028.3|:c858126-857311 SP0765 AAK74903.1 NC_003028.3|:724170-725207SP1634 AAK75714.1 NC_003028.3|:1534348-1535421 SP0418 AAK74581.1NC_003028.3|:396692-396916 SP1923 AAK75991.1NC_003028.3|:c1833311-1831896 SP1313 AAK75991.1NC_003028.3|:c1833311-1831896 SP0775 AAK74913.1NC_003028.3|:731798-732070 SP0314 AAK74491.1 NC_003028.3|:287483-290683SP0912 AAK75037.1 NC_003028.3|:864707-865465 SP0159 AAK74341.1NC_003028.3|:c157554-156292 SP0910 AAK75035.1 NC_003028.3|:863462-863734SP2148 AAK76205.1 NC_003028.3|:2062144-2063373 SP1412 AAK75510.1NC_003028.3|:c1332393-1331605 SP0372 AAK74539.1NC_003028.3|:350268-350597 SP1304 AAK75407.1NC_003028.3|:c1232491-1232390 SP2002 AAK76069.1NC_003028.3|:c1906183-1905446 SP0612 AAK74764.1NC_003028.3|:579708-579806 SP1988 AAK76055.1NC_003028.3|:c1892598-1890565 SP0484 AAK74643.1NC_003028.3|:465572-466402 SP0847 AAK74978.1 NC_003028.3|:794144-795202SP1527 AAK75616.1 NC_003028.3|:c1439494-1437536 SP0542 AAK74699.1NC_003028.3|:515940-516059 SP0441 AAK74602.1 NC_003028.3|:414869-415057SP0350 AAK74523.1 NC_003028.3|:323990-324625 SP0014 AAK74207.1NC_003028.3|:14450-14929 SP1965 AAK76032.1 NC_003028.3|:c1873279-1873073SP0117 AAK74303.1 NC_003028.3|:118423-120657 SP0981 AAK75102.1NC_003028.3|:927115-928056 SP2229 AAK76277.1NC_003028.3|:c2148627-2147602 SP2136 AAK76194.1NC_003028.3|:c2048521-2046656 SP1179 AAK75288.1NC_003028.3|:1116230-1118389 SP1174 AAK75283.1NC_003028.3|:c1110717-1108258 SP2216 AAK76264.1NC_003028.3|:c2136445-2135267 SP1393 AAK75491.1NC_003028.3|:1316756-1318027 SP0641.1 Amino acids 28-1006 Nucleotides603976-606910 of of NC_003028.3 AAK74791.1 (which is full- lengthSP0641) SP1384 AAK75482.1 NC_003028.3|:c1309464-1308967 SP2032AAK76097.1 NC_003028.3|:c1939994-1938321

Typically, the polypeptides present in compounds of the invention areimmunogenic, either alone or as a variant, which includes polypeptidesfused to another polypeptide or mixed with or complexed to an adjuvant.Variants also include sequences with less than 100% sequence identity,as described herein. In certain embodiments, an antigen of Table 1 or 2is provided as a full length polypeptide. In addition, one may usefragments, precursors and analogs that have an appropriateimmunogenicity.

These polypeptides may be immunogenic in mammals, for example mice,guinea pigs, or humans. An immunogenic polypeptide is typically onecapable of raising a significant immune response in an assay or in asubject. For instance, an immunogenic polypeptide may increase theamount of IL-17 produced by T cells. The IL-17 assay described inExamples 1-4 is an example of an assay that may be used to identify animmunogenic polypeptide. Alternatively, an immunogenic polypeptide may(i) induce production of antibodies, e.g., neutralizing antibodies, thatbind to the polypeptide (ii) induce T_(H)1 immunity, (iii) activate theCD8+ CTL response, for example by increasing CD8+ T cells and/orincreasing localization of CD8+ T cells to the site of infection orreinfection, (iv) induce T_(H)17 immunity, and/or (v) activate innateimmunity. In some embodiments, an immunogenic polypeptide causes theproduction of a detectable amount of antibody specific to that antigen.

In certain embodiments, polypeptides have less than 20%, 30%, 40%, 50%,60% or 70% identity to human autoantigens and/or gut commensal bacteria(e.g., certain Bacteroides, Clostridium, Fusobacterium, Eubacterium,Ruminococcus, Peptococcus, Peptostreptococcus, Bifidobacterium,Escherichia and Lactobacillus species). Examples of human autoantigensinclude insulin, proliferating cell nuclear antigen, cytochrome P450,and myelin basic protein.

The present disclosure provides, for example, a vaccine formulationcomprising a pharmaceutically acceptable carrier and one or morepolypeptides having an amino acid sequence comprising any of SEQ ID NOS:1-11 or an immunogenic fragment thereof, and optionally furthercomprising a polypeptide having an amino acid sequence comprising eitherof SEQ ID NOS: 12 or 13 or an immunogenic fragment thereof. In certainembodiments, the vaccine formulation comprises at least two differentpolypeptides having an amino acid sequence comprising any of SEQ ID NOS:1-13 or an immunogenic fragment thereof, wherein at least one of saidpolypeptides has an amino acid sequence comprising one of SEQ ID NOS:1-10 or an immunogenic fragment thereof. Here, the term “different”signifies that each of said two peptides originates from a differentsequence selected from SEQ ID NOS: 1-13.

The vaccine formulation may also comprise one or more polypeptideshaving an amino acid sequence consisting of any of SEQ ID NOS: 1-11.

In some embodiments, the vaccine formulation comprises at least twopolypeptides, each polypeptide belonging to a different group of(i)-(vi): (i) SEQ ID NO: 1 or an immunogenic fragment thereof, (ii) oneof SEQ ID NOS: 2-5 or an immunogenic fragment thereof, (iii) one of SEQID NOS: 6-7 or an immunogenic fragment thereof, (iv) SEQ ID NO: 8 or animmunogenic fragment thereof, (v) one of SEQ ID NOS: 9-10 or animmunogenic fragment thereof, and (vi) one of SEQ ID NO: 11-13 or animmunogenic fragment thereof. Examples of such combinations are listedbelow:

SEQ ID NO: 1 and SEQ ID NO: 2

SEQ ID NO: 1 and SEQ ID NO: 3

SEQ ID NO: 1 and SEQ ID NO: 4

SEQ ID NO: 1 and SEQ ID NO: 5

SEQ ID NO: 1 and SEQ ID NO: 6

SEQ ID NO: 1 and SEQ ID NO: 7

SEQ ID NO: 1 and SEQ ID NO: 8

SEQ ID NO: 1 and SEQ ID NO: 9

SEQ ID NO: 1 and SEQ ID NO: 10

SEQ ID NO: 1 and SEQ ID NO: 11

SEQ ID NO: 1 and SEQ ID NO: 12

SEQ ID NO: 1 and SEQ ID NO: 13

SEQ ID NO: 2 and SEQ ID NO: 6

SEQ ID NO: 2 and SEQ ID NO: 7

SEQ ID NO: 2 and SEQ ID NO: 8

SEQ ID NO: 2 and SEQ ID NO: 9

SEQ ID NO: 2 and SEQ ID NO: 10

SEQ ID NO: 2 and SEQ ID NO: 11

SEQ ID NO: 2 and SEQ ID NO: 12

SEQ ID NO: 2 and SEQ ID NO: 13

SEQ ID NO: 3 and SEQ ID NO: 6

SEQ ID NO: 3 and SEQ ID NO: 7

SEQ ID NO: 3 and SEQ ID NO: 8

SEQ ID NO: 3 and SEQ ID NO: 9

SEQ ID NO: 3 and SEQ ID NO: 10

SEQ ID NO: 3 and SEQ ID NO: 11

SEQ ID NO: 3 and SEQ ID NO: 12

SEQ ID NO: 3 and SEQ ID NO: 13

SEQ ID NO: 4 and SEQ ID NO: 6

SEQ ID NO: 4 and SEQ ID NO: 7

SEQ ID NO: 4 and SEQ ID NO: 8

SEQ ID NO: 4 and SEQ ID NO: 9

SEQ ID NO: 4 and SEQ ID NO: 10

SEQ ID NO: 4 and SEQ ID NO: 11

SEQ ID NO: 4 and SEQ ID NO: 12

SEQ ID NO: 4 and SEQ ID NO: 13

SEQ ID NO: 5 and SEQ ID NO: 6

SEQ ID NO: 5 and SEQ ID NO: 7

SEQ ID NO: 5 and SEQ ID NO: 8

SEQ ID NO: 5 and SEQ ID NO: 9

SEQ ID NO: 5 and SEQ ID NO: 10

SEQ ID NO: 5 and SEQ ID NO: 11

SEQ ID NO: 5 and SEQ ID NO: 12

SEQ ID NO: 5 and SEQ ID NO: 13

SEQ ID NO: 6 and SEQ ID NO: 8

SEQ ID NO: 6 and SEQ ID NO: 9

SEQ ID NO: 6 and SEQ ID NO: 10

SEQ ID NO: 6 and SEQ ID NO: 11

SEQ ID NO: 6 and SEQ ID NO: 12

SEQ ID NO: 6 and SEQ ID NO: 13

SEQ ID NO: 7 and SEQ ID NO: 8

SEQ ID NO: 7 and SEQ ID NO: 9

SEQ ID NO: 7 and SEQ ID NO: 10

SEQ ID NO: 7 and SEQ ID NO: 11

SEQ ID NO: 7 and SEQ ID NO: 12

SEQ ID NO: 7 and SEQ ID NO: 13

SEQ ID NO: 8 and SEQ ID NO: 9

SEQ ID NO: 8 and SEQ ID NO: 10

SEQ ID NO: 8 and SEQ ID NO: 11

SEQ ID NO: 8 and SEQ ID NO: 12

SEQ ID NO: 8 and SEQ ID NO: 13

SEQ ID NO: 9 and SEQ ID NO: 11

SEQ ID NO: 9 and SEQ ID NO: 12

SEQ ID NO: 9 and SEQ ID NO: 13

SEQ ID NO: 10 and SEQ ID NO: 11

SEQ ID NO: 10 and SEQ ID NO: 12

SEQ ID NO: 10 and SEQ ID NO: 13

In certain embodiments, the vaccine formulation comprises at least threedifferent polypeptides having an amino acid sequence comprising any ofSEQ ID NOS: 1-13 or an immunogenic fragment thereof, wherein at leastone of said polypeptides has an amino acid sequence comprising one ofSEQ ID NOS: 1-10. In certain such embodiments, the vaccine formulationcomprises at least three polypeptides, each polypeptide belonging to adifferent group of (i)-(vi): (i) SEQ ID NO: 1 or an immunogenic fragmentthereof, (ii) one of SEQ ID NOS: 2-5 or an immunogenic fragment thereof,(iii) one of SEQ ID NOS: 6-7 or an immunogenic fragment thereof, (iv)SEQ ID NO: 8 or an immunogenic fragment thereof, (v) one of SEQ ID NOS:9-10 or an immunogenic fragment thereof, and (vi) one of SEQ ID NO:11-13 or an immunogenic fragment thereof. Examples of such combinationsare listed below:

SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 6

SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 7

SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 8

SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 9

SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 10

SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 11

SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 12

SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 13

SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 6

SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 7

SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 8

SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 9

SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 10

SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 11

SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 12

SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 13

SEQ ID NO: 1, SEQ ID NO: 4; and SEQ ID NO: 6

SEQ ID NO: 1, SEQ ID NO: 4; and SEQ ID NO: 7

SEQ ID NO: 1, SEQ ID NO: 4; and SEQ ID NO: 8

SEQ ID NO: 1, SEQ ID NO: 4; and SEQ ID NO: 9

SEQ ID NO: 1, SEQ ID NO: 4; and SEQ ID NO: 10

SEQ ID NO: 1, SEQ ID NO: 4; and SEQ ID NO: 11

SEQ ID NO: 1, SEQ ID NO: 4; and SEQ ID NO: 12

SEQ ID NO: 1, SEQ ID NO: 4; and SEQ ID NO: 13

SEQ ID NO: 1, SEQ ID NO: 5; and SEQ ID NO: 6

SEQ ID NO: 1, SEQ ID NO: 5; and SEQ ID NO: 7

SEQ ID NO: 1, SEQ ID NO: 5; and SEQ ID NO: 8

SEQ ID NO: 1, SEQ ID NO: 5; and SEQ ID NO: 9

SEQ ID NO: 1, SEQ ID NO: 5; and SEQ ID NO: 10

SEQ ID NO: 1, SEQ ID NO: 5; and SEQ ID NO: 11

SEQ ID NO: 1, SEQ ID NO: 5; and SEQ ID NO: 12

SEQ ID NO: 1, SEQ ID NO: 5; and SEQ ID NO: 13

SEQ ID NO: 1, SEQ ID NO: 6; and SEQ ID NO: 8

SEQ ID NO: 1, SEQ ID NO: 6; and SEQ ID NO: 9

SEQ ID NO: 1, SEQ ID NO: 6; and SEQ ID NO: 10

SEQ ID NO: 1, SEQ ID NO: 6; and SEQ ID NO: 11

SEQ ID NO: 1, SEQ ID NO: 6; and SEQ ID NO: 12

SEQ ID NO: 1, SEQ ID NO: 6; and SEQ ID NO: 13

SEQ ID NO: 1, SEQ ID NO: 7; and SEQ ID NO: 8

SEQ ID NO: 1, SEQ ID NO: 7; and SEQ ID NO: 9

SEQ ID NO: 1, SEQ ID NO: 7; and SEQ ID NO: 10

SEQ ID NO: 1, SEQ ID NO: 7; and SEQ ID NO: 11

SEQ ID NO: 1, SEQ ID NO: 7; and SEQ ID NO: 12

SEQ ID NO: 1, SEQ ID NO: 7; and SEQ ID NO: 13

SEQ ID NO: 1, SEQ ID NO: 8; and SEQ ID NO: 9

SEQ ID NO: 1, SEQ ID NO: 8; and SEQ ID NO: 10

SEQ ID NO: 1, SEQ ID NO: 8; and SEQ ID NO: 11

SEQ ID NO: 1, SEQ ID NO: 8; and SEQ ID NO: 12

SEQ ID NO: 1, SEQ ID NO: 8; and SEQ ID NO: 13

SEQ ID NO: 1, SEQ ID NO: 9; and SEQ ID NO: 11

SEQ ID NO: 1, SEQ ID NO: 9; and SEQ ID NO: 12

SEQ ID NO: 1, SEQ ID NO: 9; and SEQ ID NO: 13

SEQ ID NO: 1, SEQ ID NO: 10; and SEQ ID NO: 11

SEQ ID NO: 1, SEQ ID NO: 10; and SEQ ID NO: 12

SEQ ID NO: 1, SEQ ID NO: 10; and SEQ ID NO: 13

SEQ ID NO: 2, SEQ ID NO: 6; and SEQ ID NO: 8

SEQ ID NO: 2, SEQ ID NO: 6; and SEQ ID NO: 9

SEQ ID NO: 2, SEQ ID NO: 6; and SEQ ID NO: 10

SEQ ID NO: 2, SEQ ID NO: 6; and SEQ ID NO: 11

SEQ ID NO: 2, SEQ ID NO: 6; and SEQ ID NO: 12

SEQ ID NO: 2, SEQ ID NO: 6; and SEQ ID NO: 13

SEQ ID NO: 2, SEQ ID NO: 7; and SEQ ID NO: 8

SEQ ID NO: 2, SEQ ID NO: 7; and SEQ ID NO: 9

SEQ ID NO: 2, SEQ ID NO: 7; and SEQ ID NO: 10

SEQ ID NO: 2, SEQ ID NO: 7; and SEQ ID NO: 11

SEQ ID NO: 2, SEQ ID NO: 7; and SEQ ID NO: 12

SEQ ID NO: 2, SEQ ID NO: 7; and SEQ ID NO: 13

SEQ ID NO: 2, SEQ ID NO: 8; and SEQ ID NO: 9

SEQ ID NO: 2, SEQ ID NO: 8; and SEQ ID NO: 10

SEQ ID NO: 2, SEQ ID NO: 8; and SEQ ID NO: 11

SEQ ID NO: 2, SEQ ID NO: 8; and SEQ ID NO: 12

SEQ ID NO: 2, SEQ ID NO: 8; and SEQ ID NO: 13

SEQ ID NO: 2, SEQ ID NO: 9; and SEQ ID NO: 11

SEQ ID NO: 2, SEQ ID NO: 9; and SEQ ID NO: 12

SEQ ID NO: 2, SEQ ID NO: 9; and SEQ ID NO: 13

SEQ ID NO: 2, SEQ ID NO: 10; and SEQ ID NO: 11

SEQ ID NO: 2, SEQ ID NO: 10; and SEQ ID NO: 12

SEQ ID NO: 2, SEQ ID NO: 10; and SEQ ID NO: 13

SEQ ID NO: 3, SEQ ID NO: 6; and SEQ ID NO: 8

SEQ ID NO: 3, SEQ ID NO: 6; and SEQ ID NO: 9

SEQ ID NO: 3, SEQ ID NO: 6; and SEQ ID NO: 10

SEQ ID NO: 3, SEQ ID NO: 6; and SEQ ID NO: 11

SEQ ID NO: 3, SEQ ID NO: 6; and SEQ ID NO: 12

SEQ ID NO: 3, SEQ ID NO: 6; and SEQ ID NO: 13

SEQ ID NO: 3, SEQ ID NO: 7; and SEQ ID NO: 8

SEQ ID NO: 3, SEQ ID NO: 7; and SEQ ID NO: 9

SEQ ID NO: 3, SEQ ID NO: 7; and SEQ ID NO: 10

SEQ ID NO: 3, SEQ ID NO: 7; and SEQ ID NO: 11

SEQ ID NO: 3, SEQ ID NO: 7; and SEQ ID NO: 12

SEQ ID NO: 3, SEQ ID NO: 7; and SEQ ID NO: 13

SEQ ID NO: 3, SEQ ID NO: 8; and SEQ ID NO: 9

SEQ ID NO: 3, SEQ ID NO: 8; and SEQ ID NO: 10

SEQ ID NO: 3, SEQ ID NO: 8; and SEQ ID NO: 11

SEQ ID NO: 3, SEQ ID NO: 8; and SEQ ID NO: 12

SEQ ID NO: 3, SEQ ID NO: 8; and SEQ ID NO: 13

SEQ ID NO: 3, SEQ ID NO: 9; and SEQ ID NO: 11

SEQ ID NO: 3, SEQ ID NO: 9; and SEQ ID NO: 12

SEQ ID NO: 3, SEQ ID NO: 9; and SEQ ID NO: 13

SEQ ID NO: 3, SEQ ID NO: 10; and SEQ ID NO: 11

SEQ ID NO: 3, SEQ ID NO: 10; and SEQ ID NO: 12

SEQ ID NO: 3, SEQ ID NO: 10; and SEQ ID NO: 13

SEQ ID NO: 4, SEQ ID NO: 6; and SEQ ID NO: 8

SEQ ID NO: 4, SEQ ID NO: 6; and SEQ ID NO: 9

SEQ ID NO: 4, SEQ ID NO: 6; and SEQ ID NO: 10

SEQ ID NO: 4, SEQ ID NO: 6; and SEQ ID NO: 11

SEQ ID NO: 4, SEQ ID NO: 6; and SEQ ID NO: 12

SEQ ID NO: 4, SEQ ID NO: 6; and SEQ ID NO: 13

SEQ ID NO: 4, SEQ ID NO: 7; and SEQ ID NO: 8

SEQ ID NO: 4, SEQ ID NO: 7; and SEQ ID NO: 9

SEQ ID NO: 4, SEQ ID NO: 7; and SEQ ID NO: 10

SEQ ID NO: 4, SEQ ID NO: 7; and SEQ ID NO: 11

SEQ ID NO: 4, SEQ ID NO: 7; and SEQ ID NO: 12

SEQ ID NO: 4, SEQ ID NO: 7; and SEQ ID NO: 13

SEQ ID NO: 4, SEQ ID NO: 8; and SEQ ID NO: 9

SEQ ID NO: 4, SEQ ID NO: 8; and SEQ ID NO: 10

SEQ ID NO: 4, SEQ ID NO: 8; and SEQ ID NO: 11

SEQ ID NO: 4, SEQ ID NO: 8; and SEQ ID NO: 12

SEQ ID NO: 4, SEQ ID NO: 8; and SEQ ID NO: 13

SEQ ID NO: 4, SEQ ID NO: 9; and SEQ ID NO: 11

SEQ ID NO: 4, SEQ ID NO: 9; and SEQ ID NO: 12

SEQ ID NO: 4, SEQ ID NO: 9; and SEQ ID NO: 13

SEQ ID NO: 4, SEQ ID NO: 10; and SEQ ID NO: 11

SEQ ID NO: 4, SEQ ID NO: 10; and SEQ ID NO: 12

SEQ ID NO: 4, SEQ ID NO: 10; and SEQ ID NO: 13

SEQ ID NO: 5, SEQ ID NO: 6; and SEQ ID NO: 8

SEQ ID NO: 5, SEQ ID NO: 6; and SEQ ID NO: 9

SEQ ID NO: 5, SEQ ID NO: 6; and SEQ ID NO: 10

SEQ ID NO: 5, SEQ ID NO: 6; and SEQ ID NO: 11

SEQ ID NO: 5, SEQ ID NO: 6; and SEQ ID NO: 12

SEQ ID NO: 5, SEQ ID NO: 6; and SEQ ID NO: 13

SEQ ID NO: 5, SEQ ID NO: 7; and SEQ ID NO: 8

SEQ ID NO: 5, SEQ ID NO: 7; and SEQ ID NO: 9

SEQ ID NO: 5, SEQ ID NO: 7; and SEQ ID NO: 10

SEQ ID NO: 5, SEQ ID NO: 7; and SEQ ID NO: 11

SEQ ID NO: 5, SEQ ID NO: 7; and SEQ ID NO: 12

SEQ ID NO: 5, SEQ ID NO: 7; and SEQ ID NO: 13

SEQ ID NO: 5, SEQ ID NO: 8; and SEQ ID NO: 9

SEQ ID NO: 5, SEQ ID NO: 8; and SEQ ID NO: 10

SEQ ID NO: 5, SEQ ID NO: 8; and SEQ ID NO: 11

SEQ ID NO: 5, SEQ ID NO: 8; and SEQ ID NO: 12

SEQ ID NO: 5, SEQ ID NO: 8; and SEQ ID NO: 13

SEQ ID NO: 5, SEQ ID NO: 9; and SEQ ID NO: 11

SEQ ID NO: 5, SEQ ID NO: 9; and SEQ ID NO: 12

SEQ ID NO: 5, SEQ ID NO: 9; and SEQ ID NO: 13

SEQ ID NO: 5, SEQ ID NO: 10; and SEQ ID NO: 11

SEQ ID NO: 5, SEQ ID NO: 10; and SEQ ID NO: 12

SEQ ID NO: 5, SEQ ID NO: 10; and SEQ ID NO: 13

SEQ ID NO: 6, SEQ ID NO: 8; and SEQ ID NO: 9

SEQ ID NO: 6, SEQ ID NO: 8; and SEQ ID NO: 10

SEQ ID NO: 6, SEQ ID NO: 8; and SEQ ID NO: 11

SEQ ID NO: 6, SEQ ID NO: 8; and SEQ ID NO: 12

SEQ ID NO: 6, SEQ ID NO: 8; and SEQ ID NO: 13

SEQ ID NO: 6, SEQ ID NO: 9; and SEQ ID NO: 11

SEQ ID NO: 6, SEQ ID NO: 9; and SEQ ID NO: 12

SEQ ID NO: 6, SEQ ID NO: 9; and SEQ ID NO: 13

SEQ ID NO: 6, SEQ ID NO: 10; and SEQ ID NO: 11

SEQ ID NO: 6, SEQ ID NO: 10; and SEQ ID NO: 12

SEQ ID NO: 6, SEQ ID NO: 10; and SEQ ID NO: 13

SEQ ID NO: 7, SEQ ID NO: 8; and SEQ ID NO: 9

SEQ ID NO: 7, SEQ ID NO: 8; and SEQ ID NO: 10

SEQ ID NO: 7, SEQ ID NO: 8; and SEQ ID NO: 11

SEQ ID NO: 7, SEQ ID NO: 8; and SEQ ID NO: 12

SEQ ID NO: 7, SEQ ID NO: 8; and SEQ ID NO: 13

SEQ ID NO: 7, SEQ ID NO: 9; and SEQ ID NO: 11

SEQ ID NO: 7, SEQ ID NO: 9; and SEQ ID NO: 12

SEQ ID NO: 7, SEQ ID NO: 9; and SEQ ID NO: 13

SEQ ID NO: 7, SEQ ID NO: 10; and SEQ ID NO: 11

SEQ ID NO: 7, SEQ ID NO: 10; and SEQ ID NO: 12

SEQ ID NO: 7, SEQ ID NO: 10; and SEQ ID NO: 13

SEQ ID NO: 8, SEQ ID NO: 9; and SEQ ID NO: 11

SEQ ID NO: 8, SEQ ID NO: 9; and SEQ ID NO: 12

SEQ ID NO: 8, SEQ ID NO: 9; and SEQ ID NO: 13

SEQ ID NO: 8, SEQ ID NO: 10; and SEQ ID NO: 11

SEQ ID NO: 8, SEQ ID NO: 10; and SEQ ID NO: 12

SEQ ID NO: 8, SEQ ID NO: 10; and SEQ ID NO: 13

In some embodiments, the vaccine formulation comprises at least twodifferent polypeptides having an amino acid sequence comprising any ofSEQ ID NOS: 14-21 or an immunogenic fragment thereof. In certain suchembodiments, the vaccine formulation comprises at least twopolypeptides, each polypeptide belonging to a different group of(i)-(iii): (i) one of SEQ ID NOS: 14-17 or an immunogenic fragmentthereof, (ii) one of SEQ ID NOS: 18-19 or an immunogenic fragmentthereof; and (iii) one of SEQ ID NOS: 20-21 or an immunogenic fragmentthereof. Examples of such combinations are listed below:

SEQ ID NO: 14 and SEQ ID NO: 18

SEQ ID NO: 14 and SEQ ID NO: 19

SEQ ID NO: 14 and SEQ ID NO: 20

SEQ ID NO: 14 and SEQ ID NO: 21

SEQ ID NO: 15 and SEQ ID NO: 18

SEQ ID NO: 15 and SEQ ID NO: 19

SEQ ID NO: 15 and SEQ ID NO: 20

SEQ ID NO: 15 and SEQ ID NO: 21

SEQ ID NO: 16 and SEQ ID NO: 18

SEQ ID NO: 16 and SEQ ID NO: 19

SEQ ID NO: 16 and SEQ ID NO: 20

SEQ ID NO: 16 and SEQ ID NO: 21

SEQ ID NO: 17 and SEQ ID NO: 18

SEQ ID NO: 17 and SEQ ID NO: 19

SEQ ID NO: 17 and SEQ ID NO: 20

SEQ ID NO: 17 and SEQ ID NO: 21

SEQ ID NO: 18 and SEQ ID NO: 20

SEQ ID NO: 18 and SEQ ID NO: 21

SEQ ID NO: 19 and SEQ ID NO: 20

SEQ ID NO: 19 and SEQ ID NO: 21

In some aspects, a vaccine formulation comprising one or more of SEQ IDNOS: 14-21 further comprises a polypeptide having an amino acid sequencecomprising any of SEQ ID NOS: 1-13.

In certain embodiments, the vaccine formulation comprises at least threedifferent polypeptides having an amino acid sequence comprising any ofSEQ ID NOS: 14-21 or an immunogenic fragment thereof. In certain suchembodiments, the vaccine formulation comprises three of (i)-(iii): (i)one of SEQ ID NOS: 14-17 or an immunogenic fragment thereof, (ii) one ofSEQ ID NOS: 18-19 or an immunogenic fragment thereof; and (iii) one ofSEQ ID NOS: 20-21 or an immunogenic fragment thereof. Examples of suchcombinations are listed below:

SEQ ID NO: 14, SEQ ID NO: 18, and SEQ ID NO: 20

SEQ ID NO: 14, SEQ ID NO: 18, and SEQ ID NO: 21

SEQ ID NO: 14, SEQ ID NO: 19, and SEQ ID NO: 20

SEQ ID NO: 14, SEQ ID NO: 19, and SEQ ID NO: 21

SEQ ID NO: 15, SEQ ID NO: 18, and SEQ ID NO: 20

SEQ ID NO: 15, SEQ ID NO: 18, and SEQ ID NO: 21

SEQ ID NO: 15, SEQ ID NO: 19, and SEQ ID NO: 20

SEQ ID NO: 15, SEQ ID NO: 19, and SEQ ID NO: 21

SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 20

SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 21

SEQ ID NO: 16, SEQ ID NO: 19, and SEQ ID NO: 20

SEQ ID NO: 16, SEQ ID NO: 19, and SEQ ID NO: 21

SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 20

SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 21

SEQ ID NO: 17, SEQ ID NO: 19, and SEQ ID NO: 20

SEQ ID NO: 17, SEQ ID NO: 19, and SEQ ID NO: 21

A polypeptide may comprise one or more immunogenic portions and one ormore non-immunogenic portions. The immunogenic portions may beidentified by various methods, including protein microarrays,ELISPOT/ELISA techniques, and/or specific assays on different deletionmutants (e.g., fragments) of the polypeptide in question. Immunogenicportions may also be identified by computer algorithms. Some suchalgorithms, like EpiMatrix (produced by EpiVax), use a computationalmatrix approach. Other computational tools for identifying antigenicepitopes include PEPVAC (Promiscuous EPitope-based VACcine, hosted byDana Farber Cancer Institute on the world wide web atimmunax.dfci.harvard.edu/PEPVAC), and MHCPred (which uses a partialleast squares approach and is hosted by The Jenner Institute on theworld wide web at www.jenner.ac.uk/MHCPred). An immunogenic fragment ofa polypeptide described herein comprises at least one immunogenicportion, as measured experimentally or identified by algorithm. Peptidesidentified by the tools described above include the following:

SP2108 SP0148 SP1634 SP0882 SP0314 Fragments Fragments FragmentsFragments Fragments (SEQ ID NOS (SEQ ID NOS (SEQ ID NOS (SEQ ID NOS(SEQ ID NOS 34-57, 58-82, 83-109, 110-130, 131-169, respectively,respectively, respectively, respectively, respectively, in order ofin order of in order of in order of in order of appearance) appearance)appearance) appearance) appearance) AIIDGPWKA ALGLVAAGV RLLDLAPQVHLDNLVLKV MLKDKIAFL VMMAPYDRV ELTGYEIEV MLEIPAHQI DLIAGRVHL SLADYTYKVSIAGINYAK AVNNLSYTK KNFFAHHPK ILLPKDYEK FLLLGAFYL VWDPAKNML TYLPAEADIKVILAGHSK EYQDQIGCL VLIDGLSQL QPLPNISQM RYNMAVNNL SFDNLVSTL YFHDGQNVFILASLGFLL APYDRVGSL DFQQIMVRL YYDLPLNEL NPDISRMIV GLSQLLPVI APAVIESLVEHTDNPTIL YFDLFFGTI IPWSENLPD FLLNHYMTV FYYTYGLLA APIAQNPNV ALEYIHHLFQFGGKGVEY MLIPNVDRA SKYAFAGE LPSDQQPYV LPLNELDIL IGLEYQDQI KLEEMAKQVTEGAGNLI YVYPLLAQG IPQGSIIGM VYFHDGQN VLKRGVYTI LADWTNFYY QGLDNLKVIDPELQKQFA MEVVKPFI KVIAGLLRK SLVMYYNKD KYLYAAPI AVYTFDAPG YLKMKEHKLTLNYEHMNK KEAGVKVTL GELTGYEI QSLTPEERE KLSPDQRIF NIGYFFFKK KSTAVLGTVNPNVLVVKK AIYAASQI RIFIYVGTE KYTDVIEKF GAKTDDTTK KLSKQFFGD LEIPAHQIFIDETYRTK KYDDSVSTI SQKFVDFLV GSPRPFIYE LLDLAPQVP DTDRSYPVV TFNQMIKELQAFKDAKVN AVNNLSYTK WQIEDKHFV YIDSSLCYY DYPETQSVF AVIESLVMY KIFDKIGVETLGRLTQLL TQFIGLEYQ TPRAINNTL DAKTAANDA MVRLSDGQF LYFDLFFGT KDTDRSYPVAPLLVNGEL YGVATIPTL YVYPLLAQG SINDLASLK LCYYHDLIA YIDHTNVAY KTAAIIDGPVVQATTSAK SINDLASLK NVFNSKESF KQNGDSYGY KAYEKEAGV TLEKLSKQF YYDLPLNELFLLNHYMTV AGNGAYVFG VAAGVLAAC QKVILAGHS FYLYNGDLS AWVIPQAVK LDNLKVIELGTDDSIIGW KSFAPLLV NMAVNNLSY TYLSFDNLV DETVVRTV FGTILDAGI YIDHTNVAYNQITAVYTF MLKDKIAFL KLRFKIKTD KLELFYETG KIAFLGSNI SVPRTSYLS FGFGLSLFSSTIRSIEQV FRKTTDNPF TVVRTVRDS STIRSIEQV DGLSQLLPV FGFGLSLFS KLVDQGEGF

SP0024 Fragments SP1072 Fragments SP0641 Fragments (SEQ ID NOS 170-(SEQ ID NOS 194- (SEQ ID NOS 228- 193, respective- 227, respective-264, respective- ly, in order of ly, in order of ly, in order ofappearance) appearance) appearance) AIVTCMDSR GIEVEKPLY AAYAPNEVVAQTFENEPF AEAHLLYRM AGDLRGKII AYVALHGQL ALLNQDNMR DEIANEVWY DDVIISGAIAPPERNYLY DNYLIYGDL FENEPFQEY AQNSYIHIL DQKEHPEKF FMQANQAYV AVASMGTALDSLTDRLKL ISQQQMGTR AYLLTKTRI EAKNKNKFV KPKTRVAIV DAAKFYHAI EGQGRNRKLLHGQLNLPL DTALEELER EIKGAGDLR LHVAQALGL EEYQGVPFI EPIAEGQYF LPLKPKTRVEFLEKIAPL EVSELKPHR MGTREIVVL EFQVLYDLL GAFFDKSKI MQLLIESPL EHVEHLKRLGDLKWDGLI QANQAYVAL ELSEVEMTR GEVEKNLEV QFMQANQAY ESPLVLNDY IHFESVEEMQLNLPLKPK GEKTPSFNV IMFIVGIFL QQMGTREIV GLCPFHGEK IPGTLNKGI REIVVLHHTIGDMPVQIV IRYQVFTFK SPLIPDDVI ITMPVTKQL ISDKGGFNW SRLHVAQAL KALLNQDNMIVSEEDFIL TEDMIRSLV KRLTKKLVL KEIGVEEAI VDVSDQDFL LTKTRISPI KIVVKDFARVSDQDFLPF LVLVYDGDK KKINFQPSL VTEDMIRSL MRAEAHLLY KLKFVYIGK NGPEDLAYLKVYYGNNYK QTEEVERAW KYWQAIRAL SEIYLMEGF LHIDNTRDF SPHQALYDM MRFKKEDLKVDKQVIEEI NESVVDNYL VEMTRNKAL NEVWYAGAA VLYDLLGQY NINDIVDGL VPFIEAVQIQYLLKDNII WYQVLAQDL SPRQQGAGL YLMEGFMDV SRSKTLGGY SSLKNTKVL TAAVILAAYWTELPAMGY

Thus, in some aspects, this application provides an immunogenic fragmentof an antigen described herein. The fragments, in some instances, areclose in size to the full-length polypeptide or the polypeptide of Table1 or 2. For example, they may lack at most one, two, three, four, five,ten, or twenty amino acids from one or both termini. In certainembodiments, the polypeptide is 100-500 amino acids in length, or150-450, or 200-400, or 250-250 amino acids in length. In someembodiments, the polypeptide is 100-200, 150-250, 200-300, 250-350,300-400, 350-450, or 400-500 amino acids in length. The fragmentsdescribed above or sub-fragments thereof (e.g., fragments of 8-50, 8-30,or 8-20 amino acid residues) preferably have one of the biologicalactivities described below, such as increasing the amount of IL-17released by at least 1.5 fold or 2 fold or more (e.g., either as anabsolute measure or relative to an immunologically inactive protein suchas ovalbumin). A fragment may be used as the polypeptide in the vaccinesdescribed herein or may be fused to another protein, protein fragment ora polypeptide.

In some embodiments, the fragment is a truncated fragment of any of SEQID NOS: 1-13 having from 1-5, 1-10, or 1-20 amino acid residues removedfrom the N-terminus, C-terminus, or both. In some such embodiments, thesame number of residues is removed from the N-terminus and theC-terminus, while in other embodiments, a different number of residuesis removed from the N-terminus compared to the C-terminus.

In certain aspects, this application provides immunogenic polypeptideswith at least 90%, 95%, 97%, 98%, 99%, or 99.5% identity to apolypeptide of Table 1 or 2. The present disclosure also provides avaccine formulation comprising a pharmaceutically acceptable carrier andone or more polypeptides having an amino acid sequence comprising asequence at least 90%, 95%, 98%, or 99% identical to any of SEQ ID NOS:1-11 or an immunogenic fragment thereof, and optionally furthercomprising a polypeptide having an amino acid sequence comprising asequence at least 90%, 95%, 98%, or 99% identical to either of SEQ IDNOS: 12 or 13 or an immunogenic fragment thereof. In certainembodiments, the vaccine formulation comprises at least two differentpolypeptides having an amino acid sequence comprising a sequence atleast 90%, 95%, 98%, or 99% identical to any of SEQ ID NOS: 1-13 or animmunogenic fragment thereof, wherein at least one of said polypeptideshas an amino acid sequence comprising a sequence at least 90%, 95%, 98%,or 99% identical to one of SEQ ID NOS: 1-10 or an immunogenic fragmentthereof.

In some embodiments, one or more, e.g. two, three, four, or morepolypeptides from Table 1 or 2 or immunogenic fragments or variantsthereof are provided in a mixture. In some embodiments, two, three,four, or more polypeptides from Table 1 or 2 or immunogenic fragments orvariants thereof are covalently bound to each other, e.g. as a fusionprotein.

In some embodiments, the vaccine formulation contains substantially noother S. pneumoniae polypeptides other than polypeptides having an aminoacid sequence comprising any of SEQ ID NOS: 1-13. In some embodiments,the vaccine formulation contains substantially no other S. pneumoniaepolypeptides other than polypeptides of Table 1. In some embodiments,the vaccine formulation contains substantially no other S. pneumoniaepolypeptides other than polypeptides of Tables 1 or 2.

In certain embodiments, vaccine formulations or immunogenic compositionscontain substantially no other S. pneumoniae polypeptides other thanpolypeptides having an amino acid sequence comprising any of SEQ ID NO:1-13. In certain such embodiments, vaccine formulations or immunogeniccompositions contain substantially no other S. pneumoniae polypeptidesother than polypeptides having an amino acid sequence consisting of anyof SEQ ID NO: 1-13. In some embodiments, vaccine formulations orimmunogenic compositions contain substantially no other S. pneumoniaepolypeptides other than polypeptides having an amino acid sequencecomprising (or consisting of) any of the amino acid sequences of thepolypeptides of Tables 1 and 2. Substantially, in this context, refersto less than 50%, less than 40%, less than 30%, less than 20%, less than10%, less than 5%, less than 3%, less than 2, or even less than 1% ofthe other S. pneumoniae polypeptides.

In certain embodiments, the vaccine composition induces a T_(H)17 cellresponse at least 1.5-fold than that induced by a control irrelevantantigen (such as the HSV-2 protein ICP47 with the gene name US12) aftercontacting T_(H)17 cells. In some embodiments, the vaccine formulationinhibits infection by S. pneumoniae in an uninfected subject. In certainembodiments, the vaccine formulation inhibits S. pneumoniae colonizationin an individual. In some embodiments, the vaccine formulation inhibitsS. pneumoniae symptoms.

In certain embodiments, this application provides nucleic acids encodingone or more of the polypeptides described above, such as DNA, RNA, or ananalog thereof. The underlying DNA sequences for the polypeptidesdescribed above may be modified in ways that do not affect the sequenceof the protein product, and such sequences are included in theinvention. For instance, the DNA sequence may be codon-optimized toimprove expression in a host such as E. coli, an insect cell line (e.g.,using the baculovirus expression system), or a mammalian (e.g., human orChinese Hamster Ovary) cell line.

In certain embodiments, this application provides nucleic acids (such asDNA, RNA, or an analog thereof) that are at least 70%, 80%, 90%, 95%,97%, 98%, 99%, or 100% identical to a gene in Table 1 or 2, or a variantor portion of said gene. In certain embodiments, the nucleic acid is600-2000, 800-1800, 1000-1600, 1200-1400 nucleotides in length. In someembodiments, the nucleic acid is 600-1600, 800-1800, or 1000-2000nucleotides in length. The nucleic acids may be used, for example, forrecombinant production of the polypeptides of Tables 1 and 2, orimmunogenic fragments thereof.

In some embodiments, the vaccine or immunogenic composition may comprisefusion proteins and/or fusion DNA constructs. The polypeptides describedherein may be used without modification. In certain embodiments, whensmaller related polypeptides are used, such as fragments or the like,and their molecular weight is less than about 5000 daltons, e.g., 1500to 5000 daltons, modification may be useful in eliciting the desiredimmune response. For example, the smaller polypeptides can be conjugatedto an appropriate immunogenic carrier such as tetanus toxoid,pneumolysis keyhole limpet hemocyanin or the like. In certainembodiments, the vaccine formulation comprises at least one lipidatedpolypeptide. Conjugation may be direct or indirect (e.g., via a linker).In other embodiments, a construct may comprise a gene or protein fromTable 1 or 2 or an immunogenic fragment or variant thereof and a tag. Atag may be N-terminal or C-terminal For instance, tags may be added tothe nucleic acid or polypeptide to facilitate purification, detection,solubility, or confer other desirable characteristics on the protein ornucleic acid. For instance, a purification tag may be a peptide,oligopeptide, or polypeptide that may be used in affinity purification.Examples include His, GST, TAP, FLAG, myc, HA, MBP, VSV-G, thioredoxin,V5, avidin, streptavidin, BCCP, Calmodulin, Nus, S tags, lipoprotein D,and β galactosidase. Particular exemplary His tags include HHHHHH (SEQID NO: 32) and MSYYHHHHHH (SEQ ID NO: 33). In other embodiments, thepolypeptide is free of tags such as protein purification tags, and ispurified by a method not relying on affinity for a purification tag. Insome embodiments, the fused portion is short. This, in some instances,the fusion protein comprises no more than 1, 2, 3, 4, 5, 10, or 20additional amino acids on one or both termini of the polypeptide ofTable 1 or 2.

B. Immunogenic Compositions

The present disclosure also provides pharmaceutical compositionscontaining immunogenic polypeptides or polynucleotides encoding theseimmunogenic polypeptides together with a pharmaceutical carrier.Antigens from S. pneumoniae were identified by screening immune cellsfrom mice infected with S. pneumonia, or from healthy human donors. Thehuman donors had presumably been exposed to S. pneumoniae at some pointduring their lifetimes, because S. pneumoniae is a very common diseaseand colonizing pathogen. Briefly, a library of S. pneumoniae antigenswas expressed in bacteria and mixed with antigen presenting cells(APCs). The APCs, in turn, presented S. pneumoniae-derived polypeptidesto lymphocytes that had been isolated from mice or from human donors.Lymphocyte responses were assayed for reactivity to S. pneumoniae. Humandonors, as well as mice immunized with S. pneumoniae, producedlymphocytes specific to S. pneumoniae antigens. Thus, the presentdisclosure contemplates compositions of the S. pneumoniae antigens thatelicit a strong immune response in immunized or infected mice or humansfor counteracting infection by S. pneumoniae.

Tables 1 and 2 list the protein sequence and corresponding nucleotidesequence for S. pneumoniae antigens identified according to thescreening methods described herein. The antigens were identified inscreens of mouse and human T cells. In the screens of mouse T cells, theidentified antigens were subjected to at least two rounds of screening:a genome-wide round to identify pools of 4 antigens that elicited animmune response, followed by a deconvolution round to individually testand identify single antigens that elicited an immune response from apool identified in the genome-wide round. In contrast, in the screens ofhuman T cells, two different sets of antigen pools were created, suchthat a polypeptide was combined with different polypeptides between thefirst and second pools. Consequently, it is possible to determine whichpolypeptides are antigens by identifying which polypeptides are inpositive pools in both the first and second sets. Table 1 lists antigens(and variants thereof) that were identified by one of the abovescreening methods, and were subsequently subjected to further testing inthe mouse model described in Examples 5-8. Thus, compositions accordingto this disclosure may include one or two or more of the genes listed inTable 1 or 2, or the corresponding gene products.

An immunogenic composition may also comprise portions of saidStreptococcus polypeptides, for example deletion mutants, truncationmutants, oligonucleotides, and peptide fragments. In some embodiments,the portions of said polypeptides are immunogenic. The immunogenicity ofa portion of a protein is readily determined using the same assays thatare used to determine the immunogenicity of the full-length protein. Insome embodiments, the portion of the polypeptide has substantially thesame immunogenicity as the full-length proteins. In some embodiments,the immunogenicity is no more than 10%, 20%, 30%, 40%, or 50% less thanthat of the full-length protein (e.g., polypeptides of Tables 1 and 2).The peptide fragments may be, for example, linear, circular, orbranched.

Some embodiments of the vaccine formulations and immunogeniccompositions described herein include an immunogenic polypeptide (e.g.,a polypeptide of Table 1 or 2) that contains a membrane translocatingsequence (MTS), to facilitate introduction of the polypeptide into themammalian cell and subsequent stimulation of the cell-mediated immuneresponse. Exemplary membrane translocating sequences include hydrophobicregion in the signal sequence of Kaposi fibroblast growth factor, theMTS of α-synuclein, β-synuclein, or γ-synuclein, the third helix of theAntennapedia homeodomain, SN50, integrin P3 h-region, HIV Tat, pAntp,PR-39, abaecin, apidaecin, Bac5, Bac7, P. berghei CS protein, and thoseMTSs described in U.S. Pat. Nos. 6,248,558, 6,432,680 and 6,248,558.

In certain embodiments, an antigen (e.g., a polypeptide of Table 1 or 2)is covalently bound to another molecule. This may, for example, increasethe half-life, solubility, bioabailability, or immunogenicity of theantigen. Molecules that may be covalently bound to the antigen include acarbohydrate, biotin, poly(ethylene glycol) (PEG), polysialic acid,N-propionylated polysialic acid, nucleic acids, polysaccharides, andPLGA. There are many different types of PEG, ranging from molecularweights of below 300 g/mol to over 10,000,000 g/mol. PEG chains can belinear, branched, or with comb or star geometries. In some embodiments,the naturally produced form of a protein is covalently bound to a moeitythat stimulates the immune system. An example of such a moeity is alipid moeity. In some instances, lipid moieties are recognized by aToll-like receptor (TLR) such as TLR2, and activate the innate immunesystem.

C. Antibodies Specific to the Proteins of Tables 1 and 2

Another aspect disclosed herein is an antibody preparation generatedagainst an antigenic composition (e.g., one of the proteins listed inTable 1 or 2 or an immunogenic fragment thereof). For instance, thisdisclosure provides combinations of two, three, four, or five antibodieseach recognizing a different protein of Table 1 or 2. Any of a varietyof antibodies are included. Such antibodies include, e.g., polyclonal,monoclonal, recombinant, humanized or partially humanized, single chain,Fab, and fragments thereof, etc. The antibodies can be of any isotype,e.g., IgG, various IgG isotypes such as IgG1, IgG2, IgG2a, IgG2b, IgG3,IgG4, etc.; and they can be from any animal species that producesantibodies, including goat, rabbit, mouse, chicken or the like. In someembodiments, Fab molecules are expressed and assembled in a geneticallytransformed host like E. coli. A lambda vector system is available thusto express a population of Fab's with a potential diversity equal to orexceeding that of subject generating the predecessor antibody. See Huseet al. (1989), Science 246, 1275-81.

D. Components of a Vaccine or Immunogenic Composition Comprising S.pneumoniae Antigens or Antibodies Recognizing the Same

In certain embodiments, the vaccine or immunogenic composition comprisesan antigen and one or more of the following: an adjuvant, stabilizer,buffer, surfactant, controlled release component, salt, preservative,and an antibody specific to said antigen.

1. Adjuvants

The vaccine formulations and immunogenic compositions described hereinmay include an adjuvant. Adjuvants can be broadly separated into twoclasses, based on their principal mechanisms of action: vaccine deliverysystems and immunostimulatory adjuvants (see, e.g., Singh et al., Curr.HIV Res. 1:309-20, 2003). Vaccine delivery systems are often particulateformulations, e.g., emulsions, microparticles, immune-stimulatingcomplexes (ISCOMs), which may be, for example, particles and/ormatrices, and liposomes. In contrast, immunostimulatory adjuvants aresometimes derived from pathogens and can represent pathogen associatedmolecular patterns (PAMP), e.g., lipopolysaccharides (LPS),monophosphoryl lipid (MPL), or CpG-containing DNA, which activate cellsof the innate immune system.

Alternatively, adjuvants may be classified as organic and inorganic.Inorganic adjuvants include alum salts such as aluminum phosphate,amorphous aluminum hydroxyphosphate sulfate, and aluminum hydroxide,which are commonly used in human vaccines. Organic adjuvants compriseorganic molecules including macromolecules. An example of an organicadjuvant is cholera toxin.

Adjuvants may also be classified by the response they induce. In someembodiments, the adjuvant induces the activation of T_(H)1 cells orT_(H)2 cells. In other embodiments, the adjuvant induces the activationof B cells. In yet other embodiments, the adjuvant induces theactivation of antigen-presenting cells. These categories are notmutually exclusive; in some cases, an adjuvant activates more than onetype of cell.

In certain embodiments, the adjuvant induces the activation of T_(H)17cells. It may promote the T_(H)17 cells to secrete IL-17. In someembodiments, an adjuvant that induces the activation of T_(H)17 cells isone that produces at least a 2-fold, and in some cases a 10-fold,experimental sample to control ratio in the following assay. In theassay, an experimenter compares the IL-17 levels secreted by twopopulations of cells: (1) cells treated with the adjuvant and apolypeptide known to induce T_(H)17 activation, and (2) cells treatedwith the adjuvant and an irrelevant (control) polypeptide. An adjuvantthat induces the activation of T_(H)17 cells may cause the cells ofpopulation (1) to produce more than 2-fold, or more than 10-fold moreIL-17 than the cells of population (2). IL-17 may be measured, forexample, by ELISA or Western blot. Certain toxins, such as cholera toxinand labile toxin (produced by enterotoxigenic E. coli, or ETEC),activate a T_(H)17 response. Thus, in some embodiments, the adjuvant isa toxin. Cholera toxin was successfully used in the mouse model toinduce protective immunity in conjunction with certain polypeptides fromTable 1 (see Examples 5-8). One form of labile toxin is produced byIntercell. Mutant derivates of labile toxin that are active as adjuvantsbut significantly less toxic can be used as well. Exemplary detoxifiedmutant derivatives of labile toxin include mutants lackingADP-ribosyltransferase activity. Particular detoxified mutantderivatives of labile toxin include LTK7 (Douce et al., “Mutants ofEscherichia coli heat-labile toxin lacking ADP-ribosyltransferaseactivity act as nontoxic, mucosal adjuvants” PNAS Vol. 92, pp.1644-1648, February 1995) and LTK63 (Williams et al., “Innate Imprintingby the Modified Heat-Labile Toxin of Escherichia coli (LTK63) ProvidesGeneric Protection against Lung Infectious Disease” The Journal ofImmunology, 2004, 173: 7435-7443), LT-G192 (Douce et al. “Geneticallydetoxified mutants of heat-labile toxin from Escherichia coli are ableto act as oral adjuvants” Infect Immun. 1999 September; 67(9):4400-6),and LTR72 (“Mucosal adjuvanticity and immunogenicity of LTR72, a novelmutant of Escherichia coli heat-labile enterotoxin with partial knockoutof ADP-ribosyltransferase activity.” J Exp Med. 1998 Apr. 6;187(7):1123-32).

In some embodiments, the adjuvant comprises a VLP (virus-like particle).One such adjuvant platform, Alphavirus replicons, induces the activationof T_(H)17 cells using alphavirus and is produced by Alphavax. Incertain embodiments of the Alphavirus replicon system, alphavirus may beengineered to express an antigen of interest, a cytokine of interest(for example, IL-17 or a cytokine that stimulates IL-17 production), orboth, and may be produced in a helper cell line. More detailedinformation may be found in U.S. Pat. Nos. 5,643,576 and 6,783,939. Insome embodiments, a vaccine formulation is administered to a patient incombination with a nucleic acid encoding a cytokine.

Certain classes of adjuvants activate toll-like receptors (TLRs) inorder to activate a T_(H)17 response. TLRs are well known proteins thatmay be found on leukocyte membranes, and recognize foreign antigens(including microbial antigens). Administering a known TLR ligandtogether with an antigen of interest (for instance, as a fusion protein)can promote the development of an immune response specific to theantigen of interest. One exemplary adjuvant that activates TLRscomprises Monophosphoryl Lipid A (MPL). Traditionally, MPL has beenproduced as a detoxified lipopolysaccharide (LPS) endotoxin obtainedfrom gram negative bacteria, such as S. minnesota. In particular,sequential acid and base hydrolysis of LPS produces an immunoactivelipid A fraction (which is MPL), and lacks the saccharide groups and allbut one of the phosphates present in LPS. A number of synthetic TLRagonists (in particular, TLR4 agonists) are disclosed in Evans J T etal. “Enhancement of antigen-specific immunity via the TLR4 ligands MPLadjuvant and Ribi.529.” Expert Rev Vaccines 2003 April; 2(2):219-29.Like MPL adjuvants, these synthetic compounds activate the innate immunesystem via TLR. Another type of TLR agonist is a synthetic phospholipiddimer, for example E6020 (Ishizaka S T et al. “E6020: a syntheticToll-like receptor 4 agonist as a vaccine adjuvant.” Expert Rev.Vaccines. 2007 October; 6(5):773-84.). Various TLR agonists (includingTLR4 agonists) have been produced and/or sold by, for example, theInfectious Disease Research Institute (IRDI), Corixa, Esai, Avanti PolarLipids, Inc., and Sigma Aldrich. Another exemplary adjuvant thatactivates TLRs comprises a mixture of MPL, Trehalose Dicoynomycolate(TDM), and dioctadecyldimethylammonium bromide (DDA). AnotherTLR-activating adjuvant is R848 (resiquimod).

In some embodiments, the adjuvant is or comprises a saponin. Typically,the saponin is a triterpene glycoside, such as those isolated from thebark of the Quillaja saponaria tree. A saponin extract from a biologicalsource can be further fractionated (e.g., by chromatography) to isolatethe portions of the extract with the best adjuvant activity and withacceptable toxicity. Typical fractions of extract from Quillajasaponaria tree used as adjuvants are known as fractions A and C.

A particular form of saponins that may be used in vaccine formulationsdescribed herein is immuno stimulating complexes (ISCOMs). ISCOMs are anart-recognized class of adjuvants, that generally comprise Quillajasaponin fractions and lipids (e.g., cholesterol and phospholipids suchas phosphatidyl choline). In certain embodiments, an ISCOM is assembledtogether with a polypeptide or nucleic acid of interest. However,different saponin fractions may be used in different ratios. Inaddition, the different saponin fractions may either exist together inthe same particles or have substantially only one fraction per particle(such that the indicated ratio of fractions A and C are generated bymixing together particles with the different fractions). In thiscontext, “substantially” refers to less than 20%, 15%, 10%, 5%, 4%, 3%,2% or even 1%. Such adjuvants may comprise fraction A and fraction Cmixed into a ratio of 70-95 A:30-5 C, such as 70 A:30 C to 75 A:5 C, 75A:5 C to 80 A:20 C, 80 A:20 C to 85 A:15 C, 85 A:15 C to 90 A:10 C, 90A:10 C to 95 A:5 C, or 95 A:5 C to 99 A:1 C.

In certain embodiments, combinations of adjuvants are used. Threeexemplary combinations of adjuvants are MPL and alum, E6020 and alum,and MPL and an ISCOM.

Adjuvants may be covalently bound to antigens. In some embodiments, theadjuvant may comprise a protein which induces inflammatory responsesthrough activation of antigen-presenting cells (APCs). In someembodiments, one or more of these proteins can be recombinantly fusedwith an antigen of choice, such that the resultant fusion moleculepromotes dendritic cell maturation, activates dendritic cells to producecytokines and chemokines, and ultimately, enhances presentation of theantigen to T cells and initiation of T cell responses (see Wu et al.,Cancer Res 2005; 65(11), pp 4947-4954). In certain embodiments, apolypeptide described herein is presented in the context of thetrivalent S. pneumoniae Pneumococcal surface adhesin A: pneumolysinderivative carrying three amino acid substitutions (W433F, D385N, andC428G) which render the molecule nontoxic but do not interfere withTLR4-mediated inflammatory properties-cell wall polysaccharide(PsaA:PdT-CPs) conjugate system described in Lu et al. (“Protectionagainst Pneumococcal colonization and fatal pneumonia by a trivalentconjugate of a fusion protein with the cell wall polysaccharide.” InfectImmun. 2009 May; 77(5):2076-83). The conjugate system is “a fusionprotein of PsaA with the pneumolysin nontoxic derivative PdT and thencoupled CPs to the fusion protein”. In some embodiments, one or morepolypeptides described herein is used in place of PsaA in the trivalentconjugate. The trivalent conjugate system typically includes alum and isusually administered parenterally. Other exemplary adjuvants that may becovalently bound to antigens comprise polysaccharides, pneumolysin,synthetic peptides, lipopeptides, and nucleic acids.

Typically, the same adjuvant or mixture of adjuvants is present in eachdose of a vaccine. Optionally, however, an adjuvant may be administeredwith the first dose of vaccine and not with subsequent doses (i.e.,booster shots). Alternatively, a strong adjuvant may be administeredwith the first dose of vaccine and a weaker adjuvant or lower dose ofthe strong adjuvant may be administered with subsequent doses. Theadjuvant can be administered before the administration of the antigen,concurrent with the administration of the antigen or after theadministration of the antigen to a subject (sometimes within 1, 2, 6, or12 hours, and sometimes within 1, 2, or 5 days). Certain adjuvants areappropriate for human patients, non-human animals, or both.

2. Additional Components of a Vaccine or Immunogenic Composition

In addition to the antigens and the adjuvants described above, a vaccineformulation or immunogenic composition may include one or moreadditional components.

In certain embodiments, the vaccine formulation or immunogeniccomposition may include one or more stabilizers such as sugars (such assucrose, glucose, or fructose), phosphate (such as sodium phosphatedibasic, potassium phosphate monobasic, dibasic potassium phosphate, ormonosodium phosphate), glutamate (such as monosodium L-glutamate),gelatin (such as processed gelatin, hydrolyzed gelatin, or porcinegelatin), amino acids (such as arginine, asparagine, histidine,L-histidine, alanine, valine, leucine, isoleucine, serine, threonine,lysine, phenylalanine, tyrosine, and the alkyl esters thereof), inosine,or sodium borate.

In certain embodiments, the vaccine formulation or immunogeniccomposition includes one or more buffers such as a mixture of sodiumbicarbonate and ascorbic acid. In some embodiments, the vaccineformulation may be administered in saline, such as phosphate bufferedsaline (PBS), or distilled water.

In certain embodiments, the vaccine formulation or immunogeniccomposition includes one or more surfactants such as polysorbate 80(Tween 80), Triton X-100, Polyethylene glycol tert-octylphenyl ethert-Octylphenoxypolyethoxyethanol4-(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol (TRITON X-100);Polyoxyethylenesorbitan monolaurate Polyethylene glycol sorbitanmonolaurate (TWEEN 20); and 4-(1,1,3,3-Tetramethylbutyl)phenol polymerwith formaldehyde and oxirane (TYLOXAPOL). A surfactant can be ionic ornonionic.

In certain embodiments, the vaccine formulation or immunogeniccomposition includes one or more salts such as sodium chloride, ammoniumchloride, calcium chloride, or potassium chloride.

In certain embodiments, a preservative is included in the vaccine orimmunogenic composition. In other embodiments, no preservative is used.A preservative is most often used in multi-dose vaccine vials, and isless often needed in single-dose vaccine vials. In certain embodiments,the preservative is 2-phenoxyethanol, methyl and propyl parabens, benzylalcohol, and/or sorbic acid.

In certain embodiments, the vaccine formulation or immunogeniccomposition is a controlled release formulation.

E. DNA Vaccines

In certain aspects, the vaccine comprises one of the nucleic acidsdisclosed herein or a nucleic acid corresponding to one of thepolypeptides described herein. When a nucleic acid vaccine isadministered to a patient, the corresponding gene product (such as adesired antigen) is produced in the patient's body. In some embodiments,nucleic acid vaccine vectors that include optimized recombinantpolynucleotides can be delivered to a mammal (including humans) toinduce a therapeutic or prophylactic immune response. The nucleic acidmay be, for example, DNA, RNA, or a synthetic nucleic acid. The nucleicacid may be single stranded or double stranded.

Nucleic acid vaccine vectors (e.g., adenoviruses, liposomes,papillomaviruses, retroviruses, etc.) can be administered directly tothe mammal for transduction of cells in vivo. The nucleic acid vaccinescan be formulated as pharmaceutical compositions for administration inany suitable manner, including parenteral administration.

In determining the effective amount of the vector to be administered inthe treatment or prophylaxis of an infection or other condition, thephysician evaluates vector toxicities, progression of the disease, andthe production of anti-vector antibodies, if any. Often, the doseequivalent of a naked nucleic acid from a vector is from about 1 μg to 1mg for a typical 70 kilogram patient, and doses of vectors used todeliver the nucleic acid are calculated to yield an equivalent amount oftherapeutic nucleic acid. Administration can be accomplished via singleor divided doses. The toxicity and therapeutic efficacy of the nucleicacid vaccine vectors can be determined using standard pharmaceuticalprocedures in cell cultures or experimental animals.

A nucleic acid vaccine can contain DNA, RNA, a modified nucleic acid, ora combination thereof. In some embodiments, the vaccine comprises one ormore cloning or expression vectors; for instance, the vaccine maycomprise a plurality of expression vectors each capable of autonomousexpression of a nucleotide coding region in a mammalian cell to produceat least one immunogenic polypeptide. An expression vector oftenincludes a eukaryotic promoter sequence, such as the nucleotide sequenceof a strong eukaryotic promoter, operably linked to one or more codingregions. The compositions and methods herein may involve the use of anyparticular eukaryotic promoter, and a wide variety are known; such as aCMV or RSV promoter. The promoter can be heterologous with respect tothe host cell. The promoter used may be a constitutive promoter.

A vector useful in the present compositions and methods can be circularor linear, single-stranded or double stranded and can be a plasmid,cosmid, or episome. In a suitable embodiment, each nucleotide codingregion is on a separate vector; however, it is to be understood that oneor more coding regions can be present on a single vector, and thesecoding regions can be under the control of a single or multiplepromoters.

Numerous plasmids may be used for the production of nucleic acidvaccines. Suitable embodiments of the nucleic acid vaccine employconstructs using the plasmids VR1012 (Vical Inc., San Diego Calif.),pCMVI.UBF3/2 (S. Johnston, University of Texas) or pcDNA3.1 (InVitrogenCorporation, Carlsbad, Calif.) as the vector. In addition, the vectorconstruct can contain immunostimulatory sequences (ISS), such asunmethylated dCpG motifs, that stimulate the animal's immune system. Thenucleic acid vaccine can also encode a fusion product containing theimmunogenic polypeptide. Plasmid DNA can also be delivered usingattenuated bacteria as delivery system, a method that is suitable forDNA vaccines that are administered orally. Bacteria are transformed withan independently replicating plasmid, which becomes released into thehost cell cytoplasm following the death of the attenuated bacterium inthe host cell.

An alternative approach to delivering the nucleic acid to an animalinvolves the use of a viral or bacterial vector. Examples of suitableviral vectors include adenovirus, polio virus, pox viruses such asvaccinia, canary pox, and fowl pox, herpes viruses, including catfishherpes virus, adenovirus-associated vector, and retroviruses. Exemplarybacterial vectors include attenuated forms of Salmonella, Shigella,Edwardsiella ictaluri, Yersinia ruckerii, and Listeria monocytogenes. Insome embodiments, the nucleic acid is a vector, such as a plasmid, thatis capable of autologous expression of the nucleotide sequence encodingthe immunogenic polypeptide.

F. Use of Vaccines

The S. pneumoniae vaccines described herein may be used for prophylacticand/or therapeutic treatment of S. pneumoniae. Accordingly, thisapplication provides a method for treating a subject suffering from orsusceptible to S. pneumoniae infection, comprising administering aneffective amount of any of the vaccine formulations described herein. Insome aspects, the method inhibits S. pneumoniae colonization in anindividual. In some aspects, the method inhibits S. pneumoniae symptoms.The subject receiving the vaccination may be a male or a female, and maybe a child or adult. In some embodiments, the subject being treated is ahuman. In other embodiments, the subject is a non-human animal.

1. Prophylactic Use

In prophylactic embodiments, the vaccine is administered to a subject toinduce an immune response that can help protect against theestablishment of S. pneumoniae, for example by protecting againstcolonization, the first and necessary step in disease. Thus, in someaspects, the method inhibits infection by S. pneumoniae in anoncolonized or uninfected subject. In another aspect, the method mayreduce the duration of colonization in an individual that is alreadycolonized.

In some embodiments, the vaccine compositions of the invention conferprotective immunity, allowing a vaccinated individual to exhibit delayedonset of symptoms or reduced severity of symptoms, as the result of hisor her exposure to the vaccine. In certain embodiments, the reduction inseverity of symptoms is at least 25%, 40%, 50%, 60%, 70%, 80% or even90%. In particular embodiments, vaccinated individuals may display nosymptoms upon contact with S. pneumoniae, do not become colonized by S.pneumoniae, or both. Protective immunity is typically achieved by one ormore of the following mechanisms: mucosal, humoral, or cellularimmunity. Mucosal immunity is primarily the result of secretory IgA(sIGA) antibodies on mucosal surfaces of the respiratory,gastrointestinal, and genitourinary tracts. The sIGA antibodies aregenerated after a series of events mediated by antigen-processing cells,B and T lymphocytes, that result in sIGA production by B lymphocytes onmucosa-lined tissues of the body. Humoral immunity is typically theresult of IgG antibodies and IgM antibodies in serum. Cellular immunitycan be achieved through cytotoxic T lymphocytes or through delayed-typehypersensitivity that involves macrophages and T lymphocytes, as well asother mechanisms involving T cells without a requirement for antibodies.In particular, cellular immunity may be mediated by T_(H)1 or T_(H)17cells.

Essentially any individual has a certain risk of becoming infected withS. pneumoniae. However, certain sub-populations have an increased riskof infection. In some embodiments, a vaccine formulation as describedherein (e.g., a composition comprising one or more polypeptides fromTable 1 or 2, or nucleic acids encoding the polypeptides, or antibodiesreactive with the polypeptides) is administered to patients that areimmunocompromised.

An immunocompromising condition arising from a medical treatment islikely to expose the individual in question to a higher risk ofinfection with S. pneumoniae. It is possible to treat an infectionprophylactically in an individual having the immunocompromised conditionbefore or during treatments known to compromise immune function. Byprophylactically treating with an antigenic composition (e.g., two ormore antigens from Table 1 or 2, or nucleic acids encoding theantigens), or with antibodies reactive to two or more antigens fromTable 1 or 2, before or during a treatment known to compromise immunefunction, it is possible to prevent a subsequent S. pneumoniae infectionor to reduce the risk of the individual contracting an infection due tothe immunocompromised condition. Should the individual contract an S.pneumoniae infection e.g., following a treatment leading to animmunocompromised condition it is also possible to treat the infectionby administering to the individual an antigen composition.

The following groups are at increased risk of pneumococcal disease orits complications, and therefore it is advantageous for subjects fallinginto one or more of these groups to receive a vaccine formulationdescribed herein: children, especially those from 1 month to 5 years oldor 2 months to 2 years old; children who are at least 2 years of agewith asplenia, splenic dysfunction or sickle-cell disease; children whoare at least 2 years of age with nephrotic syndrome, chroniccerebrospinal fluid leak, HIV infection or other conditions associatedwith immunosuppression.

In another embodiment, at least one dose of the pneumococcal antigencomposition is given to adults in the following groups at increased riskof pneumococcal disease or its complications: all persons 65 years ofage; adults with asplenia, splenic dysfunction or sickle-cell disease;adults with the following conditions: chronic cardiorespiratory disease,cirrhosis, alcoholism, chronic renal disease, nephrotic syndrome,diabetes mellitus, chronic cerebrospinal fluid leak, HIV infection, AIDSand other conditions associated with immunosuppression (Hodgkin'sdisease, lymphoma, multiple myeloma, immunosuppression for organtransplantation), individuals with cochlear implants; individuals withlong-term health problems such as heart disease and lung disease, aswell as individuals who are taking any drug or treatment that lowers thebody's resistance to infection, such as long-term steroids, certaincancer drugs, radiation therapy; Alaskan natives and certain NativeAmerican populations.

2. Therapeutic Use

In therapeutic applications, the vaccine may be administered to apatient suffering from S. pneumoniae infection, in an amount sufficientto treat the patient. Treating the patient, in this case, refers toreducing symptoms, bacterial load, or both of S. pneumoniae in aninfected individual. In some embodiments, treating the patient refers toreducing the duration of symptoms or reducing the intensity of symptoms.In some embodiments, the vaccine reduces transmissibility of S.pneumoniae from the vaccinated patient. In certain embodiments, thereductions described above are at least 25%, 30%, 40%, 50%, 60%, 70%,80% or even 90%.

In therapeutic embodiments, the vaccine is administered to an individualpost-infection. The vaccine may be administered shortly after infection,e.g. before symptoms manifest, or may be administered during or aftermanifestation of symptoms.

A therapeutic S. pneumoniae vaccine can reduce the intensity and/orduration symptoms of the various indications of S. pneumoniae infection.A S. pneumoniae infection can take many forms. In some cases, aninfected patient develops pneumonia, acute sinusitis, otitis media (earinfection), meningitis, bacteremia, sepsis, osteomyelitis, septicarthritis, endocarditis, peritonitis, pericarditis, cellulitis, or brainabscess.

3. Assaying Vaccination Efficacy

The efficacy of vaccination with the vaccines disclosed herein may bedetermined in a number of ways, in addition to the clinical outcomesdescribed above. First, one may assay IL-17 levels (particularly IL-17A)by stimulating T cells derived from the subject after vaccination. TheIL-17 levels may be compared to IL-17 levels in the same subject beforevaccination. Increased IL-17 (e.g., IL-17A) levels, such as a 1.5 fold,2-fold, 5-fold, 10-fold, 20-fold, 50-fold or 100-fold or more increase,would indicate an increased response to the vaccine. Alternatively (orin combination), one may assay neutrophils in the presence of T cells orantibodies from the patient for pneumococcal killing. Increasedpneumococcal killing, such as a 1.5 fold, 2-fold, 5-fold, 10-fold,20-fold, 50-fold or 100-fold or more increase, would indicate anincreased response to the vaccine. In addition, one may measure T_(H)17cell activation, where increased T_(H)17 cell activation, such as a 1.5fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold or 100-fold or moreincrease, correlates with an increased response to the vaccine. One mayalso measure levels of an antibody specific to the vaccine, whereincreased levels of the specific antibody, such as a 1.5 fold, 2-fold,5-fold, 10-fold, 20-fold, 50-fold or 100-fold or more increase, arecorrelated with increased vaccine efficacy. In certain embodiments, twoor more of these assays are used. For example, one may measure IL-17levels and the levels of vaccine-specific antibody. Alternatively, onemay follow epidemiological markers such as incidence of, severity of, orduration of pneumococcal infection in vaccinated individuals compared tounvaccinated individuals.

Vaccine efficacy may also be assayed in various model systems such asthe mouse model. For instance, BALB/c or C57BL/6 strains of mice may beused. After administering the test vaccine to a subject (as a singledose or multiple doses), the experimenter administers a challenge doseof S. pneumoniae. In some cases, the challenge dose is sufficient tocause S. pneumoniae colonization (especially nasal colonization) in anunvaccinated animal, and in some cases the challenge dose is sufficientto cause a high rate of lethality in unvaccinated animals. One can thenmeasure the reduction in colonization or the reduction in lethality invaccinated animals. Examples 5 and 6 show the efficacy of polypeptidesof Table 1 in inhibiting S. pneumoniae nasal colonization in the mousemodel.

G. Use of Immunogenic Compositions

1. Defense Against S. pneumoniae Infection

The immunogenic compositions of the present disclosure are designed toelicit an immune response against S. pneumoniae. Compositions describedherein (e.g., ones comprising one or more polypeptides of Table 1 or 2,or nucleic acids encoding the polypeptides) may stimulate an antibodyresponse or a cell-mediated immune response, or both, in the mammal towhich it is administered. In some embodiments, the compositionstimulates a T_(H)1-biased CD4+ T cell response, a T_(H)17-biased CD4+ Tcell response, or a CD8+ T cell response; in the case of a singlecomponent composition, the composition may stimulate an antibodyresponse, a T_(H)1-biased CD4+ T cell response, T_(H)17-biased CD4+ Tcell response, and/or a CD8+ T cell response.

In certain embodiments, the composition (e.g., one comprising one ormore polypeptides of Table 1 or 2, or nucleic acids encoding thepolypeptides, or antibodies reactive with the peptides) includes acytokine or nucleotide coding region encoding a cytokine such as IL-17,to provide additional stimulation to the immune system of the mammal. Incertain embodiments, the composition comprises a cytokine such as IL-17.

While not wishing to be bound by theory, in some embodiments a T_(H)17cell response is beneficial in mounting an immune response to thecompositions disclosed herein, e.g., ones comprising one or morepolypeptides of Table 1 or 2. In certain embodiments, an active T_(H)17response is beneficial in clearing a pneumococcal infection. Forinstance, mice lacking the IL-17A receptor show decreased whole cellvaccine-based protection from a pneumococcal challenge (Lu et al.,“Interleukin-17A mediates acquired immunity to pneumococcalcolonization.” PLoS Pathog. 2008 Sep. 19; 4(9)). Furthermore, the sameauthors showed that the response level of IL-17A was increased in micetreated with a whole-cell vaccine.

Thus, herein is provided a method of increasing IL-17 production byadministering the compositions described herein (e.g., ones comprisingone or more polypeptides of Table 1 or 2) to a subject. Furthermore,this application provides a method of activating T_(H)17 cells byadministering said compositions to a subject. In certain embodiments,increased IL-17A levels result in increased pneumococcal killing byneutrophils or neutrophil-like cells, for instance by inducingrecruitment and activation of neutrophils of neutrophil-like cells. Incertain embodiments, this pneumococcal killing is independent ofantibodies and complement. However, specific antibody production andcomplement activation may be useful additional mechanisms thatcontribute to clearing of a pneumococcal infection.

Immunogenic compositions containing immunogenic polypeptides orpolynucleotides encoding immunogenic polypeptides together with apharmaceutical carrier are also provided.

In some instances, the immunogenic composition comprises one or morenucleic acids encoding one or more polypeptides of SEQ ID NOS: 1-13,such as one or more nucleic acids selected from SEQ ID Nos. 24-31. Insome embodiments these nucleic acids are expressed in the immunizedindividual, producing the encoded S. pneumoniae antigens, and the S.pneumoniae antigens so produced can produce an immunostimulatory effectin the immunized individual.

Such a nucleic acid-containing immunostimulatory composition maycomprise, for example, an origin of replication, and a promoter thatdrives expression of one or more nucleic acids encoding one or morepolypeptides of SEQ ID NOS: 1-13. Such a composition may also comprise abacterial plasmid vector into which is inserted a promoter (sometimes astrong viral promoter), one or more nucleic acids encoding one or morepolypeptides of SEQ ID NOS: 1-13, and a polyadenylation/transcriptionaltermination sequence. In some instances, the nucleic acid is DNA.

H. Diagnostic Uses

This application provides, inter alia, a rapid, inexpensive, sensitive,and specific method for detection of S. pneumoniae in patients. In thisrespect it should be useful to all hospitals and physicians examiningand treating patients with or at risk for S. pneumoniae infection.Detection kits can be simple enough to be set up in any local hospitallaboratory, and the antibodies and antigen-binding portions thereof canreadily be made available to all hospitals treating patients with or atrisk for S. pneumoniae infection. As used herein, “patient” refers to anindividual (such as a human) that either has an S. pneumoniae infectionor has the potential to contract an S. pneumoniae infection. A patientmay be an individual (such as a human) that has an S. pneumoniaeinfection, has the potential to contract an S. pneumoniae infection, whohas recovered from S. pneumoniae infection, and/or an individual whoseinfection status is unknown.

In some embodiments, one may perform a diagnostic assay using two ormore antibodies, each of which binds one of the antigens of Table 1 and2 to detect S. pneumoniae in an individual. The instant disclosure alsoprovides a method of phenotyping biological samples from patientssuspected of having a S. pneumoniae infection: (a) obtaining abiological sample from a patient; (b) contacting the sample with two ormore S. pneumoniae-specific antibodies or antigen-binding portionsthereof under conditions that allow for binding of the antibody orantigen-binding portion to an epitope of S. pneumoniae; where bindingindicates the presence of S. pneumoniae in the sample. In someembodiments, the binding to the biological sample is compared to bindingof the same antibody to a negative control tissue, wherein if thebiological sample shows the presence of S. pneumoniae as compared to thenegative control tissue, the patient is identified as likely having a S.pneumoniae infection. In some cases, binding of one antibody indicatesthe presence of S. pneumoniae; in other cases, the binding of two ormore antibodies indicates the presence of S. pneumoniae. Theaforementioned test may be appropriately adjusted to detect otherbacterial infections, for instance by using an antibody immunoreactive ahomolog (from another bacterial species) of one of the proteinsdescribed in Table 1. In some embodiments, the antibodies raised againsta S. pneumoniae protein in Table 1 or 2 will also bind the homolog inanother Streptococcus species, especially if the homologs have a highpercentage sequence identity.

Alternatively, one may use an antigen of Table 1 or 2 to detect anti-S.pneumoniae antibodies in an individual. The instant disclosure alsoprovides a method of phenotyping biological samples from patientssuspected of having a S. pneumoniae infection: (a) obtaining abiological sample from a patient; (b) contacting the sample with two ormore S. pneumoniae-specific antigens selected from Table 1 or 2 orportions thereof under conditions that allow for binding of the antigen(or portion thereof) to any host antibodies present in the sample; wherebinding indicates the presence of anti-S. pneumoniae antibodies in thesample. In some embodiments, the binding to the biological sample iscompared to binding of the same antigen to a negative control tissue,wherein if the biological sample shows the presence of anti-S.pneumoniae antibodies as compared to the negative control tissue, thepatient is identified as likely either (1) having a S. pneumoniaeinfection, or (2) having had a S. pneumoniae infection in the past. Insome cases, detecting one antibody indicates a current or past infectionwith S. pneumoniae; in other cases, detecting two or more antibodiesindicates a current or past infection with S. pneumoniae. Theaforementioned test may be appropriately adjusted to detect otherbacterial infections, for instance by using a homolog (from anotherbacterial species (e.g., a Streptococcal species) of the proteinsdescribed in Table 1.

In some embodiments, the immune cell response of a mammalian cell may bequantified ex vivo. A method for such quantification comprisesadministering the compositions herein disclosed to a mammalian T cell exvivo, and quantifying the change in cytokine production of the mammalianT cell in response to the composition. In these methods, the cytokinemay be, for example, IL-17.

The binding of an S. pneumoniae antibody to an antigen (e.g., apolypeptide of Table 1 or 2) may be measured using any appropriatemethod. Such methods include ELISA (enzyme-linked immunosorbent assay),Western blotting, competition assay, and spot-blot. The detection stepmay be, for instance, chemiluminescent, fluorescent, or colorimetric.One suitable method for measuring antibody-protein binding is theLuminex xMAP system, where peptides are bound to a dye-containingmicrosphere. Certain systems, including the xMAP system, are amenable tomeasuring several different markers in multiplex, and could be used tomeasure levels of antibodies at once. In some embodiments, other systemsare used to assay a plurality of markers in multiplex. For example,profiling may be performed using any of the following systems: antigenmicroarrays, bead microarrays, nanobarcodes particle technology, arrayedproteins from cDNA expression libraries, protein in situ array, proteinarrays of living transformants, universal protein array, lab-on-a-chipmicrofluidics, and peptides on pins. Another type of clinical assay is achemiluminescent assay to detect antibody binding. In some such assays,including the VITROS Eci anti-HCV assay, antibodies are bound to asolid-phase support made up of microparticles in liquid suspension, anda surface fluorometer is used to quantify the enzymatic generation of afluorescent product.

In some embodiments, if the biological sample shows the presence of S.pneumoniae (e.g., by detecting one or more polypeptide of Table 1 or 2or an antibody that binds one of said polypeptides), one may administera therapeutically effective amount of the compositions and therapiesdescribed herein to the patient. The biological sample may comprise, forexample, blood, semen, urine, vaginal fluid, mucus, saliva, feces,urine, cerebrospinal fluid, or a tissue sample. In some embodiments, thebiological sample is an organ intended for transplantation. In certainembodiments, before the detection step, the biological sample is subjectto culture conditions that promote the growth of S. pneumoniae.

The diagnostic tests herein (e.g., those that detect a polypeptide ofTable 1 or 2 or an antibody that binds one of said polypeptides) may beused to detect S. pneumoniae in a variety of samples, including samplestaken from patients and samples obtained from other sources. Forexample, the diagnostic tests may be used to detect S. pneumoniae infood, drink, or ingredients for food and drink; on objects such asmedical instruments, medical devices such as cochlear implants andpacemakers, shoes, clothing, furniture including hospital furniture, anddrapes including hospital drapes; or in samples taken from theenvironment such as plant samples. In some embodiments, the tests hereinmay be performed on samples taken from animals such as agriculturalanimals (cows, pigs, chickens, goats, horses and the like), companionanimals (dogs, cats, birds, and the like), or wild animals. In certainembodiments, the tests herein may be performed on samples taken fromcell cultures such as cultures of human cells that produce a therapeuticprotein, cultures of bacteria intended to produce a useful biologicalmolecule, or cultures of cells grown for research purposes.

This disclosure also provides a method of determining the location of aS. pneumoniae infection in a patient comprising: (a) administering apharmaceutical composition comprising a labeled S. pneumoniae antibodyor antigen-binding portion thereof to the patient, and (b) detecting thelabel, wherein binding indicates a S. pneumoniae infection in aparticular location in the patient. Such a diagnostic may also comprisecomparing the levels of binding in the patient to a control. In certainembodiments, the method further comprises, if the patient has a S.pneumoniae infection, treating the infection by administering atherapeutically effective amount of a S. pneumoniae-binding antibody orantigen-binding portion thereof to the patient. In certain embodiments,the method further comprises, if the patient has a S. pneumoniaeinfection, treating the infection by administering a therapeuticallyeffective amount of a S. pneumoniae protein of Table 1, or immunogenicportion thereof, to the patient. The method may further comprisedetermining the location and/or volume of the S. pneumoniae in thepatient. This method may be used to evaluate the spread of S. pneumoniaein the patient and determine whether a localized therapy is appropriate.

In some embodiments, the anti-S. pneumoniae antibodies described hereinmay be used to make a prognosis of the course of infection. In someembodiments, the anti-S. pneumoniae antibodies herein may be detected ina sample taken from a patient. If antibodies are present at normallevels, it would indicate that the patient has raised an immune responseagainst anti-S. pneumoniae. If antibodies are absent, or present atreduced levels, it would indicate that the patient is failing to raise asufficient response against anti-S. pneumoniae, and a more aggressivetreatment would be recommended. In some embodiments, antibodies presentat reduced levels refers to antibodies that are present at less than50%, 20%, 10%, 5%, 2%, or 1% the level of antibodies typical in apatient with a normal immune system. Antibodies may be detected byaffinity for any of the antigens described herein (e.g., those in Table1 and/or 2), for example using ELISA.

In some embodiments, detection of specific S. pneumoniae antigens (e.g.,those in Table 1 and/or 2) may be used to predict the progress andsymptoms of S. pneumoniae infection in a patient. It will be understoodby one of skill in the art that the methods herein are not limited todetection of S. pneumoniae. Other embodiments include the detection ofrelated bacteria including bacteria with proteins homologous to theproteins described in Table 1 or 2. Such related bacteria include, forexample, other strains of Streptococcus.

I. Doses and Routes of Administration 1. Dosage Forms, Amounts, andTiming

The amount of antigen in each vaccine or immunogenic composition dose isselected as an effective amount, which induces a prophylactic ortherapeutic response, as described above, in either a single dose orover multiple doses. Preferably, the dose is without significant,adverse side effects in typical vaccinees. Such amount will varydepending upon which specific antigen is employed. Generally, it isexpected that a dose will comprise 1-1000 μg of protein, in someinstances 2-100 μg, for instance 4-40 μg. In some aspects, the vaccineformulation comprises 1-1000 μg of the polypeptide and 1-250 μg of theadjuvant. In some embodiments, the appropriate amount of antigen to bedelivered will depend on the age, weight, and health (e.g.immunocompromised status) of a subject. When present, typically anadjuvant will be present in amounts from 1 μg-250 μg per dose, forexample 50-150 μg, 75-125 μg or 100 μg.

In some embodiments, only one dose of the vaccine is administered toachieve the results described above. In other embodiments, following aninitial vaccination, subjects receive one or more boost vaccinations,for a total of two, three, four or five vaccinations. Advantageously,the number is three or fewer. A boost vaccination may be administered,for example, about 1 month, 2 months, 4 months, 6 months, or 12 monthsafter the initial vaccination, such that one vaccination regimeninvolves administration at 0, 0.5-2 and 4-8 months. It may beadvantageous to administer split doses of vaccines which may beadministered by the same or different routes.

The vaccines and immunogenic compositions described herein may take on avariety of dosage forms. In certain embodiments, the composition isprovided in solid or powdered (e.g., lyophilized) form; it also may beprovided in solution form. In certain embodiments, a dosage form isprovided as a dose of lyophilized composition and at least one separatesterile container of diluent.

In some embodiments, the composition will be administered in a doseescalation manner, such that successive administrations of thecomposition contain a higher concentration of composition than previousadministrations. In some embodiments, the composition will beadministered in a manner such that successive administrations of thecomposition contain a lower concentration of composition than previousadministrations.

In therapeutic applications, compositions are administered to a patientsuffering from a disease in an amount sufficient to treat the patient.Therapeutic applications of a composition described herein includereducing transmissibility, slowing disease progression, reducingbacterial viability or replication, or inhibiting the expression ofproteins required for toxicity, such as by 90%, 80%, 70%, 60%, 50%, 40%,30%, 20% or 10% of the levels at which they would occur in individualswho are not treated with the composition.

In prophylactic embodiments, compositions are administered to a human orother mammal to induce an immune response that can inhibit theestablishment of an infectious disease or other condition. In someembodiments, a composition may partially block the bacterium fromestablishing an infection.

In some embodiments, the compositions are administered in combinationwith antibiotics. This co-administration is particularly appropriatewhen the pharmaceutical composition is administered to a patient who hasrecently been exposed (or is suspected of having been recently exposed)to S. pneumoniae. Many antibiotics are used to treat pneumococcalinfections, including penicillin, amoxicillin, amoxicillin/clavulanate,cefuroxime, cefotaxime, ceftriaxone, and vancomycin. The appropriateantibiotic may be selected based on the type and severity of theinfection, as well as any known antibiotic resistance of the infection(Jacobs MR “Drug-resistant Streptococcus pneumoniae: rational antibioticchoices” Am J. Med. 1999 May 3; 106(5A):19S-25S).

2. Routes of Administration

The vaccine formulations and pharmaceutical compositions herein can bedelivered by administration to an individual, typically by systemicadministration (e.g., intravenous, intraperitoneal, intramuscular,intradermal, subcutaneous, subdermal, transdermal, intracranial,intranasal, mucosal, anal, vaginal, oral, buccal route or they can beinhaled) or they can be administered by topical application. In someembodiments, the route of administration is intramuscular. In otherembodiments, the route of administration is subcutaneous. In yet otherembodiments, the route of administration is mucosal. In certainembodiments, the route of administration is transdermal or intradermal

Certain routes of administration are particularly appropriate forvaccine formulations and immunogenic compositions comprising specifiedadjuvants. In particular, transdermal administration is one suitableroute of administration for S. pneumoniae vaccines comprising toxins(e.g. cholera toxin or labile toxin); in other embodiments, theadministration is intranasal. Vaccines formulated with Alphavirusreplicons may be administered, for example, by the intramuscular or thesubcutaneous route. Vaccines comprising Monophosphory Lipid A (MPL),Trehalose Dicoynomycolate (TDM), and dioctadecyldimethylammonium bromide(DDA) are suitable (inter alia) for intramuscular and subcutaneousadministration. A vaccine comprising resiquimod may be administeredtopically or subcutaneously, for example.

3. Formulations

The vaccine formulation or immunogenic composition may be suitable foradministration to a human patient, and vaccine or immunogeniccomposition preparation may conform to USFDA guidelines. In someembodiments, the vaccine formulation or immunogenic composition issuitable for administration to a non-human animal. In some embodiments,the vaccine or immunogenic composition is substantially free of eitherendotoxins or exotoxins. Endotoxins may include pyrogens, such aslipopolysaccharide (LPS) molecules. The vaccine or immunogeniccomposition may also be substantially free of inactive protein fragmentswhich may cause a fever or other side effects. In some embodiments, thecomposition contains less than 1%, less than 0.1%, less than 0.01%, lessthan 0.001%, or less than 0.0001% of endotoxins, exotoxins, and/orinactive protein fragments. In some embodiments, the vaccine orimmunogenic composition has lower levels of pyrogens than industrialwater, tap water, or distilled water. Other vaccine or immunogeniccomposition components may be purified using methods known in the art,such as ion-exchange chromatography, ultrafiltration, or distillation.In other embodiments, the pyrogens may be inactivated or destroyed priorto administration to a patient. Raw materials for vaccines, such aswater, buffers, salts and other chemicals may also be screened anddepyrogenated. All materials in the vaccine may be sterile, and each lotof the vaccine may be tested for sterility. Thus, in certain embodimentsthe endotoxin levels in the vaccine fall below the levels set by theUSFDA, for example 0.2 endotoxin (EU)/kg of product for an intrathecalinjectable composition; 5 EU/kg of product for a non-intrathecalinjectable composition, and 0.25-0.5 EU/mL for sterile water.

In certain embodiments, the preparation comprises less than 50%, 20%,10%, or 5% (by dry weight) contaminating protein. In certainembodiments, the desired molecule is present in the substantial absenceof other biological macromolecules, such as other proteins (particularlyother proteins which may substantially mask, diminish, confuse or alterthe characteristics of the component proteins either as purifiedpreparations or in their function in the subject reconstituted mixture).In certain embodiments, at least 80%, 90%, 95%, 99%, or 99.8% (by dryweight) of biological macromolecules of the same type present (butwater, buffers, and other small molecules, especially molecules having amolecular weight of less than 5000, can be present). In someembodiments, the vaccine or immunogenic composition comprising purifiedsubunit proteins contains less than 5%, 2%, 1%, 0.5%, 0.2%, 0.1% ofprotein from host cells in which the subunit proteins were expressed,relative to the amount of purified subunit. In some embodiments, thedesired polypeptides are substantially free of nucleic acids and/orcarbohydrates. For instance, in some embodiments, the vaccine orimmunogenic composition contains less than 5%, less than 2%, less than1%, less than 0.5%, less than 0.2%, or less than 0.1% host cell DNAand/or RNA. In certain embodiments, at least 80%, 90%, 95%, 99%, or99.8% (by dry weight) of biological macromolecules of the same type arepresent in the preparation (but water, buffers, and other smallmolecules, especially molecules having a molecular weight of less than5000, can be present).

It is preferred that the vaccine or immunogenic composition has low orno toxicity, within a reasonable risk-benefit ratio. In certainembodiments, the vaccine or immunogenic composition comprisesingredients at concentrations that are less than LD₅₀ measurements forthe animal being vaccinated. LD₅₀ measurements may be obtained in miceor other experimental model systems, and extrapolated to humans andother animals. Methods for estimating the LD₅₀ of compounds in humansand other animals are well-known in the art. A vaccine formulation orimmunogenic composition, and any component within it, might have an LD₅₀value in rats of greater than 100 g/kg, greater than 50 g/kg, greaterthan 20 g/kg, greater than 10 g/kg, greater than 5 g/kg, greater than 2g/kg, greater than 1 g/kg, greater than 500 mg/kg, greater than 200mg/kg, greater than 100 mg/kg, greater than 50 mg/kg, greater than 20mg/kg, or greater than 10 mg/kg. A vaccine formulation or immunogeniccomposition that comprises a toxin such as botulinum toxin (which can beused as an adjuvant) should contain significantly less than the LD₅₀ ofbotulinum toxin.

The formulations suitable for introduction of the vaccine formulationsor pharmaceutical composition vary according to route of administration.Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous, intramuscular,intradermal, intraperitoneal, intranasal, and subcutaneous routes,include aqueous and non-aqueous, isotonic sterile injection solutions,which can contain antioxidants, buffers, bacteriostats, and solutes thatrender the formulation isotonic with the blood of the intendedrecipient, and aqueous and non-aqueous sterile suspensions that caninclude suspending agents, solubilizers, thickening agents, stabilizers,and preservatives. The formulations can be presented in unit-dose ormulti-dose sealed containers, such as ampoules and vials.

Injection solutions and suspensions can be prepared from sterilepowders, granules, and tablets of the kind previously described. In thecase of adoptive transfer of therapeutic T cells, the cells can beadministered intravenously or parenterally.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the polypeptides or packagednucleic acids suspended in diluents, such as water, saline or PEG 400;(b) capsules, sachets or tablets, each containing a predetermined amountof the active ingredient, as liquids, solids, granules or gelatin; (c)suspensions in an appropriate liquid; and (d) suitable emulsions. Tabletforms can include one or more of lactose, sucrose, mannitol, sorbitol,calcium phosphates, corn starch, potato starch, tragacanth,microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide,croscarmello se sodium, talc, magnesium stearate, stearic acid, andother excipients, colorants, fillers, binders, diluents, bufferingagents, moistening agents, preservatives, flavoring agents, dyes,disintegrating agents, and pharmaceutically compatible carriers. Lozengeforms can comprise the active ingredient in a flavor, usually sucroseand acacia or tragacanth, as well as pastilles comprising the activeingredient in an inert base, such as gelatin and glycerin or sucrose andacacia emulsions, gels, and the like containing, in addition to theactive ingredient, carriers known in the art. The pharmaceuticalcompositions can be encapsulated, e.g., in liposomes, or in aformulation that provides for slow release of the active ingredient.

The antigens, alone or in combination with other suitable components,can be made into aerosol formulations (e.g., they can be “nebulized”) tobe administered via inhalation. Aerosol formulations can be placed intopressurized acceptable propellants, such as dichlorodifluoromethane,propane, nitrogen, and the like. Aerosol formulations can be deliveredorally or nasally.

Suitable formulations for vaginal or rectal administration include, forexample, suppositories, which consist of the polypeptides or packagednucleic acids with a suppository base. Suitable suppository basesinclude natural or synthetic triglycerides or paraffin hydrocarbons. Inaddition, it is also possible to use gelatin rectal capsules whichconsist of a combination of the polypeptides or packaged nucleic acidswith a base, including, for example, liquid triglycerides, polyethyleneglycols, and paraffin hydrocarbons.

J. Preparation and Storage of Vaccine Formulations and ImmunogenicCompositions

The S. pneumoniae vaccines and immunogenic compositions described hereinmay be produced using a variety of techniques. For example, apolypeptide may be produced using recombinant DNA technology in asuitable host cell. A suitable host cell may be bacterial, yeast,mammalian, or other type of cell. The host cell may be modified toexpress an exogenous copy of one of the relevant polypeptide genes.Typically, the gene is operably linked to appropriate regulatorysequences such as a strong promoter and a polyadenylation sequence. Insome embodiments, the promoter is inducible or repressible. Otherregulatory sequences may provide for secretion or excretion of thepolypeptide of interest or retention of the polypeptide of interest inthe cytoplasm or in the membrane, depending on how one wishes to purifythe polypeptide. The gene may be present on an extrachromosomal plasmid,or may be integrated into the host genome. One of skill in the art willrecognize that it is not necessary to use a nucleic acid 100% identicalto the naturally-occurring sequence. Rather, some alterations to thesesequences are tolerated and may be desirable. For instance, the nucleicacid may be altered to take advantage of the degeneracy of the geneticcode such that the encoded polypeptide remains the same. In someembodiments, the gene is codon-optimized to improve expression in aparticular host. The nucleic acid may be produced, for example, by PCRor by chemical synthesis.

Once a recombinant cell line has been produced, a polypeptide may beisolated from it. The isolation may be accomplished, for example, byaffinity purification techniques or by physical separation techniques(e.g., a size column).

In a further aspect of the present disclosure, there is provided amethod of manufacture comprising mixing one or more polypeptides or animmunogenic fragment or variant thereof with a carrier and/or anadjuvant.

In some embodiments, antigens for inclusion the vaccine formulations andimmunogenic compositions may be produced in cell culture. One methodcomprises providing one or more expression vectors and cloningnucleotides encoding one or more polypeptides selected from polypeptideshaving an amino acid sequence of Table 1 or Table 2, then expressing andisolating the polypeptides.

The immunogenic polypeptides described herein, and nucleic acidcompositions that express the polypeptides, can be packaged in packs,dispenser devices, and kits for administering nucleic acid compositionsto a mammal. For example, packs or dispenser devices that contain one ormore unit dosage forms are provided. Typically, instructions foradministration of the compounds will be provided with the packaging,along with a suitable indication on the label that the compound issuitable for treatment of an indicated condition, such as thosedisclosed herein.

V. EXAMPLES Example 1 Antigen Identification and Pooled Murine Screens

Each open reading frame predicted in the S. pneumoniae TIGR4 genome wascloned into an expression vector comprising a tag that is able to bepresented by the major histocompatibility complex (MHC). Each constructwas then expressed in E. coli, and full-length expression validated by asurrogate assay that identifies the tag in the context of MHC. Thescreen is described in more detail in International Application WO2010/002993. In order to facilitate screening the large library, thelibrary was pooled such that four induced library clones were present ineach well. In order to screen T cells from mice immunized against S.pneumoniae, an aliquot of the pooled library was added toperitoneal-derived macrophages. The macrophages were allowed to bind thetagged S. pneumoniae antigens via the MHC. After 2 hr at 37° C., themacrophages were washed with PBS. The macrophages were then fixed with1% paraformaldehyde for 15 min and washed extensively with PBS. 10⁵ Tcells were added to each well in 200 μL of RP-10 media. The T cells hadpreviously been isolated from mice that had been immunized 2 times withkilled S. pneumoniae bacteria with cholera toxin adjuvant. The assayplates were incubated for 72 hrs at 37° C. The amount of IL-17 in thesupernatant of each well was determined through the use of an IL-17ELISA assay. The threshold for a positive result was set at two standarddeviations above the mean of all samples.

Example 2 Deconvolution of the Positive Murine Pools

A secondary screen was used to determine which antigen(s) out of thefour clones in each well induced the positive response observed in thepooled screen described in Example 1. All the clones in each positivepool were pulsed individually onto peritoneal macrophages in duplicatewells. T cells isolated from immunized mice from the same geneticbackground as the initial screen were used to screen the pulsedmacrophages using the IL-17 assay described in Example 1. Individualantigens that induced an average response in the duplicate wells greaterthan two standard deviations above the mean of negative control sampleswere considered positive responses. The library plasmids present inthese positive clones were sequenced to confirm the identity of theantigen. The antigens SP_(—)1574, SP_(—)1655, SP_(—)2106, SP_(—)0148,SP_(—)1473, SP_(—)0605, SP_(—)1177, SP_(—)0335, SP_(—)0906, SP_(—)1828,SP_(—)2157, SP_(—)1229, SP_(—)1128, SP_(—)1836, SP_(—)1865, SP_(—)0904,SP_(—)0882, SP_(—)0765, SP_(—)1634, SP_(—)0418, SP_(—)1923, SP_(—)1313,SP_(—)0775, SP_(—)0314, SP_(—)0912, SP_(—)0159, SP_(—)0910, SP_(—)2148,SP_(—)1412, SP_(—)0372, SP_(—)1304, SP_(—)2002, SP_(—)0612, SP_(—)1988,SP_(—)0484, SP_(—)0847, SP_(—)1527, SP_(—)0542, SP_(—)0441, SP_(—)0350,SP_(—)0014, SP_(—)1965, SP_(—)0117, and SP_(—)2108 were confirmed usingthis method.

Example 3 Antigen Identification and Pooled Human Screens

CD4+ T cells and CD14+ monocytes were isolated from peripheral bloodacquired from human donors. The monocytes were differentiated intodendritic cells by culturing them in GM-CSF and IL-4 containing media,essentially as described in Tedder T F and Jansen P J (1997 “Isolationand generation of human dendritic cells.” Current Protocols inImmunology Supp 23: 7.32.1-7.32.16). After five days in culture, thedendritic cells were seeded into 384 well plates. The CD4+ T cells wereexpanded in culture to ensure sufficient quantities.

Each open reading frame predicted in the S. pneumoniae TIGR4 genome wascloned into an expression vector comprising a tag that is able to bepresented by the major histocompatibility complex (MHC). Each constructwas then expressed in E. coli, and full-length expression validated by asurrogate assay that identifies the tag in the context of MHC. In orderto facilitate screening the large library, the library was pooled suchthat four induced library clones were present in each well. In order toscreen the human T cells, an aliquot of the pooled library was added tothe seeded dendritic cells in 384-well plates. After 2 hr at 37° C., thedendritic cells were fixed with 1% paraformaldehyde for 15 min andwashed extensively with phosphate buffer and lysine buffer. 40,000 ofthe CD4+ T cells in 70 μL of RP-10 media were added to each well of a384-well plate. The assay plates were incubated for 3 days at 37° C. Theamount of IL-17 in the supernatant of each well was determined throughthe use of an IL-17 ELISA assay. In different iterations of the screen,the threshold for a positive result was set at two standard deviationsabove the mean of all samples, two standard deviations above the mean ofnegative controls, or 1.78 times the median absolution deviation of thedata set. Positive pools were then deconvoluted as described in Example4.

Example 4 Deconvolution of the Positive Human Pools

For all antigens, deconvolution was performed by comparing the resultsof two pool screens. In this method, two different sets of pools wereprepared, so that a polypeptide was with three different polypeptidesbetween the first and second pools. Consequently, it is possible todetermine which polypeptides are antigens by identifying whichpolypeptides are in positive pools in both the first and second sets. Inthis deconvolution method, a pool was identified as positive if it wasat least 1.78 times the median absolution deviation of the data set.

An antigen was identified as a positive hit if it was positive in atleast two repeated secondary screens. The antigens SP2108, SP0641,SP1393, SP0024, SP0641.1, SP1072, SP1384 and SP2032 were identifiedusing the above approach.

Example 5 SP2108, SP0148 and SP1634 Polypeptides

The SP2108 polypeptide (SEQ ID NO: 9), SP0148 polypeptide (SEQ ID NO: 7)and SP1634 polypeptide (see Table 2) were formulated as vaccinecompositions using 4 μg of the polypeptide in combination with 1 μgcholera toxin. For combinations, 4 μg of each polypeptide was used. Thecompositions were administered intranasally to C57BL/6 mice three times,one week apart. The subjects were then allowed to rest for 3 weeks, andbled at that time for immunogenicity. For this assay, heparinized wholeblood was collected from the retrograde orbital sinus. The total PBMCwere stimulated with either killed whole cells (WCC) or a combination ofthe three polypeptides in round bottomed tubes for three days. Thesupernatants were then harvested and evaluated by ELISA for IL-17levels. Cholera toxin alone (CT) or an unrelated antigen from HSV (003)were used as negative controls. Results are shown in FIGS. 1 and 2. Thesubjects were allowed to rest an additional 2 weeks, at which time theywere challenged with intranasal administration of S. pneumoniae. Thesubjects were sacrificed a week later, and the number of colony-formingunits (CFU) was counted from nasal washes. Results are shown in FIG. 3.

Example 6 SP0882 and SP0314 Polypeptides

This example used the same protocols as Example 5, except that only twodoses of the vaccine composition were administered. In theseexperiments, the SP0882 polypeptide (SEQ ID NO: 2) and SP0314polypeptides (see Table 2) were used in conjunction with the threepolypeptides tested in Example 5. Results of the immunogenicity assayare shown in FIGS. 4 and 5. Results of the colonization assay are shownin FIG. 6.

Example 7 SP1072, SP0641N, and SP0024 Polypeptides

This example used a protocol similar to that of Example 5, except thattwo doses of the vaccine composition were administered, one week apart.Four weeks after the last immunization, the mice were challengedintranasally with live type 6B S. pneumoniae. One week later thebacterial burden was assessed in each mouse by plating a nasal lavage onselective media and counting CFU. The CFU isolated from each mouse isplotted for each immunized cohort. The results are shown in FIG. 7.Statistically significant results are indicated in the figure(*=p=value<0.05).

Example 8 SP0148, SP0314, SP0882, and SP2108 Polypeptides Tested in theBALB/c Mouse

To determine whether similar immune responses were seen across differentmouse genotypes, several polypeptides were administered to BALB/c mice.Using a protocol similar to that of Example 5, the mice were immunized,challenged with S. pneumoniae, and the number of CFU was recorded. Theresults of this experiment are shown in FIG. 8.

Sequences

Polypeptide Sequences SEQ ID NO: 1 SP0024 >gi|14971488|gb|AAK74215.1|conserved hypothet- ical protein Streptococcus pneumoniae TIGR4MSYFEQFMQANQAYVALHGQLNLPLKPKTRVAIVTCMDSRLHVAQALGLALGDAHILRNAGGRVTEDMIRSLVISQQQMGTREIVVLHHTDCGAQTFENEPFQEYLKEELGVDVSDQDFLPFQDIEESVREDMQLLIESPLIPD DVIISGAIYNVDTGSMTVVELSEQ ID NO: 2 SP0882 >gi|14972356|gb|AAK75009.1| conserved hypothet-ical protein (Streptococcus pneumoniae TIGR4)MNQSYFYLKMKEHKLKVPYTGKERRVRILLPKDYEKDTDRSYPVVYFHDGQNVFNSKESFIGHSWKIIPAIKRNPDISRMIVVAIDNDGMGRMNEYAAWKFQESPIPGQQFGGKGVEYAEFVMEVVKPFIDETYRTKADCQHTAMIGSSLGGNITQFIGLEYQDQIGCLGVFSSANWLHQEAFNRYFECQKLSPDQRIFIYVGTEEADDTDKTLMDGNIKQAYIDSSLCYYHDLIAGGVHLDNLVLKVQSGAIHSEIPWSENLPDCLRFFAEKW SEQ ID NO: 3 SP0882NMNQSYFYLKMKEHKLKVPYTGKERRVRILLPKDYEKDTDRSYPVVYFHDGQNVFNSKESFIGHSWKIIPAIKRNPDISRMIVVAIDNDGMGRMNEYAAWKFQESPIPGQQFGGKGVEYAEFVMEVVKPFI SEQ ID NO: 4SP0882 with exogenous leaderMSSKFMKSAAVLGTATLASLLLVACMNQSYFYLKMKEHKLKVPYTGKERRVRILLPKDYEKDTDRSYPVVYFHDGQNVFNSKESFIGHSWKIIPAIKRNPDISRMIVVAIDNDGMGRMNEYAAWKFQESPIPGQQFGGKGVEYA EFVMEVVKPF

1. A vaccine formulation comprising a pharmaceutically acceptablecarrier and one or more polypeptides having an amino acid sequencecomprising any of SEQ ID NOS: 1-11 or an immunogenic fragment thereof,and optionally further comprising a polypeptide having an amino acidsequence comprising either of SEQ ID NOS: 12 or 13 or an immunogenicfragment thereof.
 2. The vaccine formulation of claim 1, wherein thevaccine formulation comprises at least two different polypeptides havingan amino acid sequence comprising any of SEQ ID NOS: 1-13 or animmunogenic fragment thereof, wherein at least one of said polypeptideshas an amino acid sequence comprising one of SEQ ID NOS: 1-10 or animmunogenic fragment thereof.
 3. The vaccine formulation of claim 2,which comprises at least two polypeptides, each polypeptide belonging toa different group of (i)-(vi): (i) SEQ ID NO: 1 or an immunogenicfragment thereof, (ii) one of SEQ ID NOS: 2-5 or an immunogenic fragmentthereof, (iii) one of SEQ ID NOS: 6-7 or an immunogenic fragmentthereof, (iv) SEQ ID NO: 8 or an immunogenic fragment thereof, (v) oneof SEQ ID NOS: 9-10 or an immunogenic fragment thereof, and (vi) one ofSEQ ID NO: 11-13 or an immunogenic fragment thereof.
 4. The vaccineformulation of claim 1, wherein the vaccine formulation comprises atleast three different polypeptides having an amino acid sequencecomprising any of SEQ ID NOS: 1-13 or an immunogenic fragment thereof,wherein at least one of said polypeptides has an amino acid sequencecomprising one of SEQ ID NOS: 1-10.
 5. The vaccine formulation of claim4, which comprises at least three polypeptides, each polypeptidebelonging to a different group of (i)-(vi): (i) SEQ ID NO: 1 or animmunogenic fragment thereof, (ii) one of SEQ ID NOS: 2-5 or animmunogenic fragment thereof, (iii) one of SEQ ID NOS: 6-7 or animmunogenic fragment thereof, (iv) SEQ ID NO: 8 or an immunogenicfragment thereof, (v) one of SEQ ID NOS: 9-10 or an immunogenic fragmentthereof, and (vi) one of SEQ ID NO: 11-13 or an immunogenic fragmentthereof.
 6. The vaccine formulation of any of claims 1-5, wherein thefragment is a truncated fragment of any of SEQ ID NOS: 1-13 having from1-20 amino acid residues removed from the N-terminus, C-terminus, orboth.
 7. The vaccine formulation of claim 1, wherein the vaccineformulation comprises one or more polypeptides having an amino acidsequence consisting of any of SEQ ID NOS: 1-11.
 8. The vaccineformulation of claim 1, which comprises a polypeptide having an aminoacid sequence comprising SEQ ID NO:
 6. 9. The vaccine formulation ofclaim 1, which comprises a polypeptide having an amino acid sequencecomprising SEQ ID NO:
 7. 10. The vaccine formulation of claim 1, whichcomprises a polypeptide having an amino acid sequence comprising SEQ IDNO:
 9. 11. The vaccine formulation of claim 1, which comprises apolypeptide having an amino acid sequence comprising SEQ ID NO:
 10. 12.The vaccine formulation of claim 1, wherein the vaccine formulationcomprises a polypeptide consisting of SEQ ID NO: 6 and a polypeptideconsisting of SEQ ID NO:
 9. 13. The vaccine formulation of claim 1,wherein the vaccine formulation comprises a polypeptide consisting ofSEQ ID NO: 7 and a polypeptide consisting of SEQ ID NO:
 10. 14. Thevaccine formulation of any of claims 1-13, which contains substantiallyno other S. pneumoniae polypeptides other than polypeptides having anamino acid sequence comprising any of SEQ ID NOS: 1-13.
 15. The vaccineformulation of claim 1, which comprises a pharmaceutically acceptablecarrier and one or more polypeptides having an amino acid sequencecomprising any of SEQ ID NOS: 2, 7, 9, 22, and 23 or an immunogenicfragment thereof.
 16. A vaccine formulation comprising apharmaceutically acceptable carrier and a polypeptide having an aminoacid sequence consisting of SEQ ID NO: 11 or an immunogenic fragmentthereof.
 17. A vaccine formulation comprising a pharmaceuticallyacceptable carrier and a polypeptide having an amino acid sequencecomprising SEQ ID NO:
 12. 18. A vaccine formulation comprising apharmaceutically acceptable carrier and one or more polypeptides havingan amino acid sequence comprising any of SEQ ID NOS: 14-21 or animmunogenic fragment thereof.
 19. The vaccine formulation of claim 18,wherein the vaccine formulation comprises at least two differentpolypeptides having an amino acid sequence comprising any of SEQ ID NOS:14-21 or an immunogenic fragment thereof.
 20. The vaccine formulation ofclaim 19, which comprises at least two polypeptides, each polypeptidebelonging to a different group of (i)-(iii): (i) one of SEQ ID NOS:14-17 or an immunogenic fragment thereof, (ii) one of SEQ ID NOS: 18-19or an immunogenic fragment thereof; and (iii) one of SEQ ID NOS: 20-21or an immunogenic fragment thereof.
 21. The vaccine formulation of claim18, wherein the vaccine formulation further comprises a polypeptidehaving an amino acid sequence comprising any of SEQ ID NOS: 1-13. 22.The vaccine formulation of claim 18, wherein the fragment is a truncatedfragment of any of SEQ ID NOS: 14-21 wherein from 1-20 amino acidresidues are removed from the N-terminus, C-terminus, or both.
 23. Thevaccine formulation of claim 18, which comprises a polypeptide having anamino acid sequence comprising any of SEQ ID NOS: 14-17.
 24. The vaccineformulation of claim 18, which comprises a polypeptide having an aminoacid sequence comprising either of SEQ ID NOS: 18-19.
 25. The vaccineformulation of claim 18, which comprises a polypeptide having an aminoacid sequence comprising either of SEQ ID NOS: 20-21.
 26. The vaccineformulation of any of claims 1-25, wherein the polypeptide is conjugatedto an immunogenic carrier.
 27. The vaccine formulation of any of claims1-25, which comprises at least one lipidated polypeptide.
 28. Thevaccine formulation of any of claims 1-27, further comprising anadjuvant.
 29. The vaccine formulation of claim 28, wherein the adjuvantis an agonist of toll-like receptors (TLRs).
 30. The vaccine formulationof claim 28, wherein the adjuvant is alum.
 31. The vaccine formulationof claim 28, wherein the vaccine formulation comprises 1-1000 μg of thepolypeptide and 1-250 μg of the adjuvant.
 32. The vaccine formulation ofany of claims 1-31, which induces a T_(H)17 cell response at least1.5-fold after contacting T_(H)17 cells.
 33. The vaccine formulation ofany of claims 1-31, wherein the vaccine formulation inhibits infectionby S. pneumoniae in an uninfected subject.
 34. The vaccine formulationof any of claims 1-31, wherein the vaccine formulation inhibits S.pneumoniae colonization in an individual.
 35. The vaccine formulation ofany of claims 1-31, wherein the vaccine formulation inhibits S.pneumoniae symptoms.
 36. A method for treating a subject suffering fromor susceptible to S. pneumoniae infection, comprising administering aneffective amount of a vaccine formulation according to any of claims1-45.
 37. The method of claim 36, wherein the method inhibits infectionby S. pneumoniae in an uninfected subject.
 38. The method of claim 36,wherein the method inhibits S. pneumoniae colonization in an individual.39. The method of claim 36, wherein the method inhibits S. pneumoniaesymptoms.
 40. The method of claim 36, wherein the method treats asubject with one dose.
 41. The method of claim 36, wherein the methodtreats a subject within three doses.
 42. The method of claim 36, whereinthe subject is a human.
 43. An immunogenic composition comprising apharmaceutically acceptable carrier and two or more polypeptides havingamino acid sequences comprising any of SEQ ID NOS: 1-23 and SP1574,SP1655, SP2106, SP1473, SP0605, SP1177, SP0335, SP0906, SP1828, SP2157,SP1229, SP1128, SP1836, SP1865, SP0904, SP0765, SP1634, SP0418, SP1923,SP1313, SP0775, SP0314, SP0912, SP0159, SP0910, SP2148, SP1412, SP0372,SP1304, SP2002, SP0612, SP1988, SP0484, SP0847, SP1527, SP0542, SP0441,SP0350, SP0014, SP1965, SP0117, SP0981, SP2229, SP2136, SP1179, SP1174,SP2216, SP1393, SP0641.1, SP1384, and SP2032, or an immunogenic fragmentthereof.